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WO2024205094A1 - Humanized antibodies targeting epha10 - Google Patents

Humanized antibodies targeting epha10 Download PDF

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Publication number
WO2024205094A1
WO2024205094A1 PCT/KR2024/003367 KR2024003367W WO2024205094A1 WO 2024205094 A1 WO2024205094 A1 WO 2024205094A1 KR 2024003367 W KR2024003367 W KR 2024003367W WO 2024205094 A1 WO2024205094 A1 WO 2024205094A1
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Prior art keywords
antibody
cancer
epha10
fragment
cell
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PCT/KR2024/003367
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French (fr)
Korean (ko)
Inventor
정상택
이지선
한가람
차종호
신지수
김민지
유태현
이상우
이수현
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Ajou University Industry Academic Cooperation Foundation
Korea University Research and Business Foundation
Inha University Research and Business Foundation
Original Assignee
Ajou University Industry Academic Cooperation Foundation
Korea University Research and Business Foundation
Inha University Research and Business Foundation
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Application filed by Ajou University Industry Academic Cooperation Foundation, Korea University Research and Business Foundation, Inha University Research and Business Foundation filed Critical Ajou University Industry Academic Cooperation Foundation
Priority to CN202480021716.1A priority Critical patent/CN120981483A/en
Publication of WO2024205094A1 publication Critical patent/WO2024205094A1/en
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/33—Antibodies; T-cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/422—Ephrin Receptors [Eph]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to humanized antibodies targeting human EphA10 and uses thereof.
  • Cancer is a condition in which cell proliferation and death are not normally controlled due to various changes in gene expression, resulting in abnormal cell growth, which infiltrates and destroys adjacent tissues and metastasizes to other parts, ultimately leading to death.
  • the exact cause of cancer i.e. the mechanism through which normal cells are transformed into cancer cells, has not been identified, but according to what is currently known, cancer is known to be caused by a complex intertwining of external factors such as environmental factors, chemicals, radiation, and viruses, and internal factors such as genetic factors and immunological factors.
  • Cancer is broadly classified into blood cancer, which shows abnormalities in the number of blood cells, and solid cancer, which is a lump of cells with a certain hardness and shape in the body.
  • Cancer can occur in almost any part of the blood, tissue, and body, and examples include lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer.
  • the main methods of cancer treatment are surgery, radiation therapy, and anticancer treatment using chemotherapy agents that suppress cell proliferation.
  • Medicines for cancer treatment are largely divided into small molecule drugs and large molecule drugs. Compared to small molecule drugs, which have relatively more side effects due to lack of specificity, large molecule drugs with higher specificity are receiving attention as treatment agents.
  • protein therapeutics show very high specificity for disease targets and low side effects and toxicity, they are rapidly replacing non-specific small molecule compound therapeutics and are widely used in clinical settings.
  • antibody therapeutics and Fc-fusion protein therapeutics that are fused with antibody Fc regions are the main types.
  • Therapeutic antibodies show very high specificity for their targets compared to existing small molecule drugs, and are considered one of the most effective cancer treatments because they not only have low biotoxicity and few side effects, but also have an excellent blood half-life of about 3 weeks.
  • large pharmaceutical companies and research institutes around the world are spurring research and development of therapeutic antibodies that specifically bind to cancer cells, including carcinogenic factors, and effectively remove them.
  • Therapeutic antibody drug development companies include pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS.
  • Roche is leading the global antibody drug market, generating huge profits with its representative products such as Herceptin, Avastin, and Rituxan for anticancer treatment, achieving sales of approximately 19.5 billion dollars in the global market in 2012 with these three therapeutic antibodies.
  • Johnson & Johnson which developed Remicade, is also rapidly growing in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have many therapeutic antibodies in the final stages of development.
  • biopharmaceuticals including therapeutic antibodies that are specific to disease targets and have low side effects are quickly taking their place.
  • Immune checkpoint inhibitors are drugs that attack cancer cells by blocking the activity of immune checkpoint proteins involved in T cell suppression and activating T cells.
  • Representative examples use antibodies that recognize CTLA 4, PD-1, and PD-L1.
  • the response rate of cancer patients to immunotherapy including immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies, and anticancer vaccines, still remains at the level of 15 to 45%, and most solid tumors have the problem of acquiring nonresponsiveness and resistance to immunotherapy.
  • EphA10 is a type of Ephrin receptor, the largest family of RTKs (receptor tyrosine kinases). Unlike other Ephrin receptors, EphA10 is a very novel receptor whose function has been rarely studied.
  • TNBC Multiple Negative Breast Cancer
  • the purpose of the present invention is to provide an antibody specific for EphA10 or a fragment thereof having immunological activity.
  • CAR chimeric antigen receptor
  • the present invention provides an antibody specific for EphA10 or a fragment thereof having immunological activity.
  • the present invention provides an antibody-drug conjugate specific for EphA10 comprising the antibody or a fragment thereof having immunological activity.
  • the present invention also provides a bispecific or multispecific antibody comprising the antibody or a fragment having immunological activity thereof.
  • the present invention also provides a chimeric antigen receptor comprising the antibody or a fragment thereof having immunological activity.
  • the present invention provides a chimeric antigen receptor expressing cell.
  • the present invention provides a T cell engager comprising the antibody or a fragment thereof having immunological activity.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer.
  • the present invention provides a composition for cancer diagnosis.
  • the present invention also provides a use of the antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager for the prevention or treatment of cancer.
  • the present invention also provides a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager.
  • the present invention provides a use of the antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager for the diagnosis of cancer.
  • EphA10 of the present invention specifically binds to EphA10, which is a very potent target for overcoming the non-responsiveness and resistance of existing immune checkpoint inhibitor targeted therapeutics, and when an ADC is produced using the antibody, it was shown to specifically kill cancer cells. Therefore, it can be used as an antibody therapeutic for treating cancer utilizing a cancer cell death mechanism by immune cell activation, an antibody therapeutic for treating EphA10-expressing cancer, or a cell therapeutic for treating EphA10-expressing cancer, and it also has an effect that can be used for the diagnosis of cancer.
  • Figure 1 is a diagram showing the size and purity of 10 EphA10 antigen proteins expressed and purified in animal cells using SDS-PAGE gel.
  • Figure 2 shows the results of analyzing the amino acid sequence of the EphA10 target mouse antibody and the results of analyzing the binding ability of the chimeric #2 antibody produced based on the amino acid sequence to EphA10.
  • Figure 3 is a diagram showing the amino acid sequence and antibody information of an EphA10 targeting humanized antibody.
  • Figure 4 is a diagram showing the results of analyzing the expression levels of EphA10 targeting humanized antibodies expressed and purified in animal cells and confirming the size and purity of the purified humanized antibodies using an SDS-PAGE gel.
  • Figure 5 shows the results of ELISA analysis of the binding affinity of five EphA10-targeting humanized antibodies to EphA10.
  • Figure 6 is a diagram confirming the binding affinity of five types of EphA10-targeting humanized antibodies to EphA10 expressed in human and mouse cells.
  • FIG. 7 shows the binding of EphA10 targeting humanized antibodies to EphA10. This diagram shows the results of quantitative binding constant analysis.
  • Figure 8 shows the results of analyzing the antibody-drug binding of ADCs manufactured with EphA10-specific antibodies (1-1 and 1-2) selected for their high binding affinity.
  • Figure 9 is a diagram confirming the cancer cell killing efficacy of ADC produced with EphA10 specific antibodies (1-1 and 1-2).
  • Figure 10 is a diagram showing the sequence of an EphA10-specific humanized antibody, identifying free cysteine residues present in the light chain.
  • Figure 11 is a diagram showing the results of ELISA analysis of the binding affinity of EphA10-specific humanized antibody (1-1) and EphA10-targeting ADC antibody (1-1 (ADC)) produced using the same to EphA10:
  • EphA10 specific humanized antibody EphA10 specific humanized antibody
  • ADC EphA10 targeting ADC antibody.
  • Figure 12 is a diagram showing the size and purity of EphA10-specific humanized antibodies with improved ADC efficacy, in which the free cysteine residues present in the light chains of EphA10-specific antibodies 1-1 and 1-2 were substituted with alanine (A) or serine (S), respectively, expressed and purified in animal cells, and confirmed by SDS-PAGE gel.
  • A alanine
  • S serine
  • Figure 13 shows the results of analyzing the binding affinity of EphA10-specific humanized antibodies with improved ADC efficacy to EphA10.
  • Figure 14 is a diagram illustrating a yeast library construction and screening strategy for increasing the binding affinity of humanized antibodies targeting EphA10.
  • Figure 15 is a diagram showing an iterative screening process using a flow cytometer to increase the binding affinity of humanized antibodies targeting EphA10.
  • Figure 16 shows the amino acid sequence analysis results of 10 antibodies (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) discovered through exploration using a flow cytometer and 6 additional humanized antibodies (1-R, 1-W, 1-Y, 2-N, 2-R, and 2-Y) with free cysteines substituted.
  • Figure 17 is a diagram showing the size and purity of 16 humanized antibodies with increased binding affinity to EphA10 expressed and purified in animal cells, confirmed by SDS-PAGE gel.
  • Figure 18 shows the results of analyzing the binding affinity to EphA10 of 16 humanized antibodies with increased binding affinity to EphA10.
  • Figure 19 shows the results of analyzing the antibody-drug binding of ADCs produced with 2 types (A7 and 2W) among 16 humanized antibodies with increased binding affinity to EphA10.
  • Figure 20 is a diagram showing the results of ELISA analysis of the binding affinity to EphA10 of EphA10-specific antibody 1-1 and ADC manufactured using the same (1-1_ADC), humanized antibodies A7 and 2W with increased binding affinity to EphA10, and EphA10-targeting ADC antibodies manufactured using the same (A7_ADC and 2W_ADC).
  • Figure 21 is a diagram confirming the cancer cell killing efficacy of A7_ADC and 2W_ADC.
  • Figure 22 is a diagram showing the results of quantitative binding constant analysis of humanized antibodies A7 and 2W with increased binding affinity to EphA10 for EphA10.
  • Figure 23 shows the size and purity of a fusion protein containing four mouse EphA10 antigens expressed and purified in animal cells, confirmed by SDS-PAGE gel, and its activity analyzed by ELISA.
  • Figure 24 is a diagram showing the results of ELISA analysis of the binding affinity of humanized antibodies A7 and 2W with increased binding affinity to EphA10 to mouse EphA10.
  • amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
  • the present invention relates to an antibody specific for EphA10 (Eph receptor A10) or a fragment thereof having immunological activity.
  • EphA10 can be human EphA10 or mouse EphA10.
  • the antibody or immunologically active fragment thereof of the present invention can specifically bind to the extra cellular domain (ECD) of EphA10 or the FNIII domain of the extra cellular domain.
  • ECD extra cellular domain
  • the antibody of the present invention or an immunologically active fragment thereof may comprise a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 3, CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 4 to 6, and CDRH3 comprising an amino acid sequence of SEQ ID NOs: 7 or 8.
  • CDRH Complementarity determining regions Heavy chain
  • the antibody of the present invention or an immunologically active fragment thereof can comprise a VL domain comprising CDRL1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 9 to 14, CDRL2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 or 16, and CDRL3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19.
  • the antibody of the present invention or an immunologically active fragment thereof may comprise a VH domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 20 to 25.
  • the antibody of the present invention or an immunologically active fragment thereof may comprise a VL domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 26 to 45.
  • antibody 1-1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.
  • antibody 1-2 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 27.
  • antibody A.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 21 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.
  • antibody A.7 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 29.
  • antibody A.8 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30.
  • antibody B.1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 22 and a VL domain comprising the amino acid sequence of SEQ ID NO: 31.
  • antibody B.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32.
  • antibody B.7 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 24 and a VL domain comprising the amino acid sequence of SEQ ID NO: 33.
  • antibody 2.1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34.
  • antibody 2.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 35.
  • antibody 1-A of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.
  • antibody 1-N of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 37.
  • antibody 1-R of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.
  • antibody 1-W of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 39.
  • antibody 1-Y of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 40.
  • antibody 2-A of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 41.
  • antibody 2-W of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 42.
  • antibody 2-N of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 43.
  • antibody 2-R of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44.
  • antibody 2-Y of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 45.
  • the antibody or immunologically active fragment thereof of the present invention may comprise a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof of human origin.
  • the fragment having immunological activity can be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody, more preferably a humanized antibody, Fab or scFv.
  • the antibody of the invention may be a monoclonal antibody.
  • the above antibody is in the form of a whole antibody as well as a functional fragment of an antibody molecule.
  • a whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond.
  • a functional fragment of an antibody molecule means a fragment having an antigen-binding function
  • examples of antibody fragments include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a first constant region (CH1) of a heavy chain; (ii) a Fd fragment consisting of a VH and a CH1 domain; (iii) an Fv fragment consisting of a VL and a VH domain of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) joined by a peptide linker that joins the V
  • the antibody of the present invention or a fragment thereof having immunological activity may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof having immunological activity.
  • the antibody may be produced recombinantly or synthetically.
  • Animal-derived antibodies produced by immunizing a desired antigen to an immunized animal can generally cause an immune rejection reaction when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection reactions.
  • Chimeric antibodies are produced by replacing the constant region of an animal-derived antibody, which causes an anti-isotype reaction, with a constant region of a human antibody using genetic engineering methods. Although chimeric antibodies have significantly improved the anti-isotype reaction compared to animal-derived antibodies, they still contain animal-derived amino acids in the variable region, which carries the side effect of a potential anti-idiotypic reaction. Humanized antibodies have been developed to improve such side effects. These are produced by grafting the CDR (complementarity determining regions), which play an important role in antigen binding among the variable regions of chimeric antibodies, onto a human antibody framework.
  • CDR complementarity determining regions
  • the most important thing in the CDR grafting technology for producing humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody.
  • antibody databases, crystal structure analysis, and molecular modeling technology are utilized.
  • the application of additional antibody engineering technology to restore antigen binding ability can be said to be essential.
  • the above antibody or fragment having immunological activity may be isolated from a living organism (not existing in the organism) or non-naturally occurring, for example, may be synthetically or recombinantly produced.
  • the term "antibody” means a substance produced by antigen stimulation in the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly).
  • Antibodies are very stable not only in vitro but also in vivo and have a long half-life, so they are advantageous for mass expression and production.
  • antibodies inherently have a dimer structure, so they have very high avidity.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond.
  • the constant region of antibodies is divided into the heavy chain constant region and the light chain constant region.
  • the heavy chain constant region has the gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types, and the subclasses are gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1), and alpha 2 ( ⁇ 2).
  • the constant region of the light chain has the kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • the term "heavy chain” is interpreted to mean a full-length heavy chain and fragments thereof, including a variable region domain V H including an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains C H1 , C H2 and C H3 and a hinge.
  • the term "light chain” is interpreted to mean a full-length light chain and fragments thereof, including a variable region domain V L including an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain C L .
  • variable region or variable domain means a portion of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen
  • variable region includes complementarity determining regions, CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 Between the CDRs, a framework region (FR) portion exists that supports the CDR ring.
  • FR framework region
  • the "complementarity determining region” is a ring-shaped portion involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this portion changes.
  • the term "scFv (single chain fragment variable)" refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in the form of a single chain in which the VH region and the VL region of an antibody are connected by a short peptide chain.
  • the term “scFv” is intended to include scFv fragments including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This will be apparent to those skilled in the art.
  • CDR complementarity determining region
  • the heavy chain and the light chain may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3).
  • the CDRs may provide key contact residues for binding of an antibody to an antigen or an epitope.
  • the terms “specifically bind” or “specifically recognize” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.
  • the term "antigen-binding fragment” refers to a fragment of the entire structure of an immunoglobulin, and means a part of a polypeptide including a portion capable of binding an antigen.
  • it may be scFv, (scFv) 2 , scFv-Fc, Fab, Fab' or F(ab') 2 , but is not limited thereto.
  • Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain, and a first constant region (C H1 ) of a heavy chain, and has one antigen-binding site.
  • Fab' differs from Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain C H1 domain.
  • F(ab') 2 antibody is produced when the cysteine residues in the hinge region of Fab' form a disulfide bond.
  • Fv is the minimum antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant technology for producing Fv fragments is widely known in the art.
  • a two-chain Fv has a heavy chain variable region and a light chain variable region linked by a non-covalent bond
  • a single-chain Fv generally has a heavy chain variable region and a single chain variable region linked by a covalent bond or directly at the C-terminus via a peptide linker to form a dimer structure like a two-chain Fv.
  • the linker may be a peptide linker composed of any amino acid of 1 to 100 or 2 to 50, and suitable sequences are known in the art.
  • the antigen-binding fragment can be obtained using a protein hydrolase (for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and a F(ab') 2 fragment can be obtained by digestion with pepsin), or can be produced by a genetic recombination technique.
  • a protein hydrolase for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and a F(ab') 2 fragment can be obtained by digestion with pepsin
  • hinge region refers to a region included in the heavy chain of an antibody, which exists between the C H1 and C H2 regions and has the function of providing flexibility to the antigen binding site within the antibody.
  • the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, for example, IgG1, IgG2, IgG 3, or IgG4.
  • amino acid modification/mutation means substitution, insertion and/or deletion, preferably substitution, of an amino acid in a polypeptide sequence.
  • amino acid substitution or “substitution” means that an amino acid at a specific position in a polypeptide sequence of an antibody is replaced with another amino acid.
  • the antibody of the present invention or a fragment thereof having immunological activity can be prepared by any method known in the art. After encoding the polypeptide sequence of the antibody of the present invention or a fragment thereof having immunological activity, it is used to form a nucleic acid that is cloned into a host cell and expressed and assayed, if desired. Various methods for this are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).
  • the nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity can be inserted into an expression vector for protein expression.
  • the expression vector typically includes a protein operably linked, i.e., in a functional relationship, with a regulatory sequence, a selectable marker, an optional fusion partner, and/or additional elements.
  • the antibody according to the present invention or a fragment thereof having immunological activity can be produced by a method of culturing a host cell transformed with the nucleic acid, preferably an expression vector containing a nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity, to induce protein expression.
  • host cells including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used.
  • the antibody according to the present invention or a fragment thereof having immunological activity is produced using E. coli, which has low production costs and high industrial utility, as a host cell.
  • the scope of the present invention includes a method for producing an antibody or a fragment having immunological activity specific for EphA10, comprising the steps of: culturing a host cell into which a nucleic acid encoding the antibody or a fragment having immunological activity thereof of the present invention has been introduced under conditions suitable for protein expression; and purifying or isolating the antibody or fragment having immunological activity thereof expressed from the host cell.
  • Antibodies can be isolated or purified by a variety of methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunohistochemistry, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, a variety of natural proteins bind antibodies, such as, for example, bacterial proteins A, G, and L, and such proteins can be used for purification. Often, purification by specific fusion partners can be achieved.
  • the present invention relates to an antibody-drug conjugate (ADC), comprising an antibody specific for EphA10 of the present invention or a fragment thereof having immunological activity, and a drug.
  • ADC antibody-drug conjugate
  • the drug may be an immunogenic apoptosis inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a radionuclide, or an anticancer agent.
  • the pro-apoptotic peptide can be selected from the group consisting of KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-Magainin 2 (CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, Gomesin, Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin, and combinations thereof.
  • the immunogenic apoptosis inducer can be selected from the group consisting of anthracycline anticancer agent, taxane anticancer agent, anti-EGFR antibody, BK channel agonist, bortezomib, cardiac glycoside, cyclophosphamide anticancer agent, GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.
  • the anticancer agent is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duo
  • the anticancer agent may be an immunotherapy agent
  • the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant control drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.
  • the immunosuppressant controlling drug may be a drug that reduces the level of regulatory T cells (Treg), and thereby activates the immune response of T effector cells.
  • the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR, and can be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a variant thereof.
  • the drug is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin,
  • the antibody-drug conjugate may be connected/bound to the antibody of the present invention or a fragment thereof having immunological activity and the drug via a linker
  • the linker may be any linker having a functional group capable of binding to an amine group, a carboxyl group, or a sulfhydryl group of a protein such as a peptide, a ligand, an antibody, or an antibody fragment, or a phosphate group, a hydroxyl group of a nucleic acid such as an aptamer.
  • the functional group of such a linker may be isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide,
  • the linker may be an imidoester, a carbodiimide, an anhydride, a fluorophenyl ester, a hydroxymethyl phosphine, a maleimide, a haloacetyl, a pyridyldisulfide, a thiosulfonate, or a vinylsulfone.
  • the linker may be a linker that is cleavable by a protease, a linker that is cleavable under acid or base conditions, a linker that is cleavable by high temperature or light irradiation, a linker that is cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions.
  • Cleavable linkers include, for example, a hydrazone linker that is cleavable under acidic conditions, a peptide linker that is cleavable by a protease, a linker having a disulfide functional group that is cleavable under reducing conditions, and a non-cleavable linker includes, for example, Examples of the linker include MCC (Maleimidomethyl cyclohexane-1-carboxylate), MC (maleimidocaproyl) linker, or derivatives thereof such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linker or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).
  • MCC Micromediamidomethyl cyclohexane-1-carboxylate
  • MC maleimidoc
  • linker may be a self-immolative linker or a traceless linker.
  • Self-immolative linkers include, for example, linkers disclosed in U.S. Pat. No. 9,089,614 entitled "Hydrophilic self-immolative linkers and conjugates thereof," linkers disclosed in International Publication No.
  • WO2015038426 entitled “SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF,” and traceless linkers include, for example, phenylhydrazide linkers, aryl-triazene linkers, and linkers disclosed in the literature [Blaney, et al., “Traceless solid-phase organic synthesis,” Chem Rev. It may be a linker, etc. disclosed in [102: 2607-2024 (2002)].
  • the antibody of the present invention or a fragment thereof having immunological activity and the drug of the antibody-drug conjugate of the present invention may be combined via a biocompatible polymer or a carrier.
  • a biocompatible polymer means a polymer having tissue compatibility and blood compatibility that does not cause necrosis of tissue or coagulation of blood upon contact with living tissue or blood.
  • Synthetic polymers as the biocompatible polymers include polyesters, polyhydroxyalkanoates (PHAs), poly( ⁇ -hydroxyacids), poly( ⁇ -hydroxyacids), poly(3-hydroxybutyrate-co-valerate; PHBV), poly(3-hydroxyproprionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxyacids), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycolide; PLGA), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), Polycyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), poly(tyrosine carbonate), poly
  • the antibody-drug conjugate can further comprise an ADC linker, which can be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).
  • ADC linker can be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit
  • the antibody-drug conjugate can form a complex with an antibody specific for EphA10 of the present invention or a fragment thereof having immunological activity, through an ADC linker.
  • an antibody specific for EphA10 of the present invention or an immunologically active fragment thereof may be additionally conjugated to an RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule (see Shoari et al., Pharmaceutics 13:1391, pp. 1-32 (2021)).
  • the present invention relates to a bispecific or multispecific antibody comprising an antibody specific for EphA10 of the present invention or a fragment having immunological activity thereof, and a portion that binds to a target antigen other than EphA10.
  • the moiety that binds to the target antigen may comprise an antibody or a fragment having immunological activity thereof.
  • the target antigen is an antigen specific for 17-1A antigen, GD3 ganglioside R24, EGFRvIII, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrantly glycosylated MUC1, fibronectin isoform containing ED-B domain, HER2/neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, A33 antibody, Ba 733, BrE3-antigen, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD
  • the apoptosis-related genes are ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, It can be PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWH
  • the oncogene is SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1 , ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D , CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC
  • the target antigen can be a cell surface antigen or an autoantigen.
  • the cell surface antigen can be at least one selected from the group consisting of CEA, ED-B fibronectin, CD20, CD22, CDl9, EGFR, IGFlR, VEFGRl/Flt-1, VEGFR2/KDR, VEGRF3/Flt-4, HER2/neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fiber knob, PfMSP-1, HN/NDV, EpCAM/17-lA, hTR, IL-2R/Tac, CA19-9, MUCl, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, Ml3 coat protein, and GPIIb/IIIa.
  • the present invention relates to an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment having immunological activity thereof, or a bispecific or multispecific antibody of the present invention, a vector comprising the same, and a host cell transformed therewith.
  • the nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences.
  • An isolated nucleic acid is a nucleic acid that is separated from the surrounding genetic sequence present in the genome of the organism from which the nucleic acid is isolated, in the case of a nucleic acid isolated from a naturally occurring source.
  • the nucleic acid resulting from such a procedure may be understood to be an isolated nucleic acid molecule.
  • An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
  • a nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • the DNA of a pre-sequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein, that is, a form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned so as to facilitate translation.
  • Operably linked generally means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in a conventional manner.
  • the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention may have various modifications in the coding region within a range that does not change the amino acid sequence of the antibody expressed from the coding region due to the degeneracy of the codon or in consideration of the codon preferred in the organism to which the antibody is to be expressed, and various modifications or alterations may be made in a portion excluding the coding region within a range that does not affect the expression of the gene, and it will be well understood by those skilled in the art that such modified genes are also included in the scope of the present invention.
  • the nucleic acid molecule of the present invention may have one or more nucleic acid bases mutated by substitution, deletion, insertion, or a combination thereof, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention.
  • the sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
  • the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention can be inserted into an expression vector for protein expression.
  • the expression vector typically comprises the protein operably linked, i.e., in a functional relationship, with a regulatory or control sequence, a selectable marker, an optional fusion partner, and/or additional elements.
  • the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention can be produced by a method of culturing a host cell transformed with the nucleic acid, preferably an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention, to induce protein expression.
  • a host cell transformed with the nucleic acid, preferably an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention, to induce protein expression.
  • suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used.
  • E. coli which has a low production cost and high industrial utility value, can be used as the host cell
  • the vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like.
  • a suitable vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be variously prepared depending on the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc.
  • the vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, an origin of replication.
  • vector means a carrier into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence.
  • the nucleic acid sequence may be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages).
  • expression control sequences such as promoter, terminator, enhancer, etc., sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be produced.
  • expression vector means a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, a polypeptide, or a peptide.
  • Expression vectors may contain various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.
  • the term "host cell” in the present invention includes eukaryotes and prokaryotes, and means any transformable organism capable of replicating the vector or expressing a gene encoded by the vector.
  • the host cell can be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is transferred or introduced into the host cell.
  • the host cell can be a bacterial or an animal cell
  • the animal cell line can be a CHO cell, a HEK cell or a NSO cell
  • the bacteria can be Escherichia coli.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease or condition selected from the group consisting of autoimmune diseases, neurodegenerative diseases, Alzheimer's disease, metabolic diseases, cardiovascular diseases, atherosclerosis, organ transplant rejection, diseases or symptoms caused by fungi, viruses, bacteria or parasites, comprising an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, or a bispecific or multispecific antibody of the present invention as an active ingredient.
  • a disease or condition selected from the group consisting of autoimmune diseases, neurodegenerative diseases, Alzheimer's disease, metabolic diseases, cardiovascular diseases, atherosclerosis, organ transplant rejection, diseases or symptoms caused by fungi, viruses, bacteria or parasites, comprising an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, or a bispecific or multispecific antibody of the present invention as an active ingredient.
  • the present invention relates to a chimeric antigen receptor (CAR) comprising an antibody of the present invention or a fragment having immunological activity thereof as an antigen binding domain.
  • CAR chimeric antigen receptor
  • the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.
  • the present invention relates to a chimeric antigen receptor expressing cell transformed with the recombinant vector.
  • the chimeric antigen receptor expressing cell can be a chimeric antigen receptor expressing macrophage (CAR-macrophage), a chimeric antigen receptor expressing T (CAR-T) cell, a chimeric antigen receptor expressing-gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.
  • CAR-macrophage a chimeric antigen receptor expressing macrophage
  • CAR-T chimeric antigen receptor expressing T
  • CAR-Gamma-delta T a chimeric antigen receptor expressing-gamma-delta T
  • CAR-NK natural killer
  • CAR chimeric antigen receptor
  • the term "CAR (chimeric antigen receptor)” refers to a non-naturally occurring receptor capable of imparting specificity for a specific antigen to immune effector cells.
  • the CAR refers to a receptor used to transplant the specificity of a monoclonal antibody into T cells.
  • CAR is usually composed of an extracellular domain (Ectodomain), a transmembrane domain, and an intracellular domain (Ectodomain).
  • the above extracellular domain includes an antigen recognition region, and the transmembrane domain of the CAR is in a form connected to the extracellular domain, and may be derived from natural or synthetic sources. If derived from a naturally existing source, it may be derived from a membrane-bound or membrane-permeable protein, and may be a portion derived from a membrane-permeable region of various proteins such as the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154 or CD8.
  • the sequence of such a transmembrane domain can be obtained from, but is not limited to, documents known in the art that disclose membrane-permeable region portions of membrane-permeable proteins.
  • transmembrane domain when the above-mentioned transmembrane domain is synthetic, it may mainly include hydrophobic amino acid residues such as leucine and valine, and for example, a triplet of phenylalanine, tryptophan and valine may be present in the synthetic transmembrane domain, but is not limited thereto. Sequence information on such transmembrane domain can be obtained from literature known in the art regarding synthetic transmembrane domains, but is not limited thereto.
  • the intracellular domain is a part of the domain of the CAR existing within a cell, and is connected to the transmembrane domain.
  • the intracellular domain of the present invention may include an intracellular signaling domain which is characterized by causing T cell activation, preferably T cell proliferation, when an antigen binds to the antigen binding site of the CAR.
  • the intracellular signaling domain is not particularly limited in its type as long as it is a part that transmits a signal capable of causing T cell activation when an antibody binds to the antigen binding site existing outside the cell, and various types of intracellular signaling domains may be used.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM includes, but is not limited to, those derived from CD3 zeta ( ⁇ , zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d or Fc ⁇ RI ⁇ .
  • CARs comprise a single-chain fragment variable region (scFv) of an antibody specific for a tumor-associated antigen (TAA) that is coupled via the hinge and transmembrane regions to the cytoplasmic domain of a T-cell signaling molecule.
  • Most common lymphocyte activating moieties comprise a T-cell costimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domain in tandem with a T-cell triggering (e.g., CD3 ⁇ ) moiety.
  • TAA-associated antigen e.g., CD28, CD137, OX40, ICOS, and CD27
  • the term "chimeric antigen receptor expressing T (CAR-T) cell” refers to a T cell expressing a CAR.
  • the chimeric antigen receptor expressing T (CAR-T) cell has the advantage of: i) recognizing a cancer antigen in a manner independent of HLA (human leukocyte antigen), and thus capable of treating cancers that evade the action of anticancer agents by reducing HLA expression on the cell surface; ii) being independent of the HLA type, and thus capable of being used for treatment regardless of the patient's HLA type; and iii) capable of producing a large amount of cancer-specific T cells in a short period of time, and thus capable of exhibiting an excellent anticancer effect.
  • HLA human leukocyte antigen
  • T cells include CD4 + T cells (helper T cells, TH cells), CD8 + T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc., and the T cells into which the CAR is introduced in the present invention are preferably CD8 + T cells, but are not limited thereto.
  • the invention relates to a T-cell engager comprising an antibody or a fragment having immunological activity thereof.
  • the T cell engager can be, for example, a bispecific T-cell engager (BiTE).
  • the BiTE is a class of artificial bispecific monoclonal antibodies, which are fusion proteins consisting of amino acid sequences from four different genes or two single chain variable region fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons.
  • scFv single chain variable region fragments
  • the BiTE forms a link between the T cell and the tumor cell. It causes the T cell to exert cytotoxic activity on the tumor cell independently of the presence of MHC I or costimulatory molecules by producing proteins such as perforin and granzymes. These proteins enter the tumor cell and initiate apoptosis of the cell.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an antibody of the present invention or a fragment thereof having immunological activity, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell or a T cell engager.
  • the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem It may be any one selected from the group consisting of
  • the pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy, and when such combination therapy is performed, cancer can be treated more effectively.
  • chemotherapeutic agents that can be used together with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, etc.
  • Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and ⁇ -ray ir
  • the composition of the present invention may further comprise an immunogenic apoptosis inducer, wherein the immunogenic apoptosis inducer may be at least one selected from the group consisting of an anthracycline series anticancer agent, a taxane series anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide series anticancer agent, a GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline series anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or It could be valrubicin, and the taxane family of chemotherapy agents could be paclitaxel or docetaxel.
  • the pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the cancer cell killing effect by being administered together with a chemical anticancer drug (anticancer agent), etc.
  • the combined administration can be performed simultaneously with or sequentially with the anticancer agent.
  • anticancer agent examples include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin;
  • Anti-cancer antibiotics include, but are not limited to, dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan.
  • prevention means any act of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.
  • treatment means all acts that kill cancer cells or improve or beneficially change the symptoms of cancer by administering the composition of the present invention.
  • anyone with ordinary knowledge in the technical field to which the present invention belongs will be able to know the exact criteria for diseases to which the composition of the present invention is effective and determine the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association, etc.
  • the term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage when used in humans should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations when determining the effective amount are described in, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined according to factors including the patient's health condition, the type and severity of cancer, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment period, the drug used in combination or simultaneously, and other factors well known in the medical field.
  • the composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive.
  • the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc.
  • the pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.
  • composition of the present invention may also include a carrier, a diluent, an excipient or a combination of two or more thereof commonly used in biological preparations.
  • the pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary.
  • a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule or a tablet.
  • a main-use dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule or a tablet.
  • it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • composition of the present invention can be administered non-orally (for example, intravenously, subcutaneously, intraperitoneally, or locally as an injection formulation) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
  • the daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer once a day or in divided doses several times.
  • Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.
  • the present invention relates to an anticancer adjuvant comprising as an active ingredient an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager.
  • the anticancer adjuvant of the present invention may be administered concurrently, separately, or sequentially with an immunotherapy agent.
  • the anticancer adjuvant of the present invention can improve responsivity to immunotherapy.
  • the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant modulating drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.
  • the immunosuppressant controlling agent may be a drug that reduces the level of regulatory T cells (Treg).
  • the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR.
  • the present invention relates to a composition for diagnosing cancer, comprising as an active ingredient an antibody of the present invention or a fragment thereof having immunological activity, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager.
  • the label can be a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe or a tag
  • the fluorescent substance can be a fluorescent substance of the Cy(cyanine) series, Rhodamine series, Alexa series, BODIPY series or ROX series, and can be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine or Alexa.
  • the present invention relates to a method for producing an antibody or an immunologically active fragment thereof specific for EphA10, comprising the steps of: a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof; and b) recovering the polypeptide expressed by the host cell.
  • the present invention relates to a process for producing a bispecific or multispecific antibody, comprising the steps of: a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding a bispecific or multispecific antibody of the present invention; and b) recovering the polypeptide expressed by the host cell.
  • purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using Protein A.
  • the present invention relates to the use of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention for the prevention or treatment of cancer.
  • the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention.
  • the present invention relates to the use of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention for the diagnosis of cancer.
  • EphA10 Eph receptor A10
  • ECD extra cellular domain
  • an animal cell expression vector for the ECD or FNIII domain of EphA10 was produced, and for more effective antibody screening by binding activity (avidity), an animal cell expression vector for a dimeric EphA10 protein fused with GST (Glutathione-S-transferase) or human Fc, respectively, and an animal cell expression vector for expressing a tetrameric EphA10 protein fused with streptavidin were also produced.
  • Expi293F cells were sub-cultured at a density of 2 ⁇ 10 6 cells/ml, and one day later, each vector constructed above was transfected using PEI (Polyethylenimine, Polyscience, 23966).
  • EphA10-specific mouse antibody m#2 was secured through mouse immunization and hybridoma technology (Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495-497), and based on this, chimeric #2 antibody with human constant region introduced was secured through animal cell expression and antibody purification (Fig. 2).
  • EphA10-specific humanized antibodies 1-1, 1-2, 2-1, and 2-2
  • four types of EphA10-specific humanized antibodies (1-1, 1-2, 2-1, and 2-2) were secured by introducing the frameworks of two human heavy chains (IGHV2-5*09, IGHV4-38-2*02) and light chains (IGKV2-30*02, IGKV2-29*02) that have the highest similarity to the EphA10-specific mouse antibody, respectively.
  • one humanized antibody (TRA #2) that introduced the framework of trastuzumab a humanized antibody with excellent physical properties among therapeutic antibodies currently widely used in clinical practice, was also additionally produced (Fig. 3).
  • Expi293F cells were sub-cultured at a density of 2 ⁇ 10 6 cells/ml, and one day later, transfected with the heavy and light chain expression vectors produced above into PEI (Polyethylenimine, Polyscience, 23966), and then cultured with shaking in a CO 2 incubator at 37 °C, 125 rpm, and 8% CO 2 for 7 days.
  • PEI Polyethylenimine, Polyscience, 23966
  • EphA10-binding humanized antibodies purified in Example 3 to EphA10 was analyzed by ELISA. Specifically, 4 ⁇ g/ml of EphA10 fused to human Fc was diluted with 0.05 M Na 2 CO 3 (pH 9.6) solution, 50 ⁇ l each was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 ⁇ l of 4% skim milk (GenomicBase) at room temperature for 2 hours.
  • EphA10-binding humanized antibodies purified in Example 3 above was analyzed, and antibodies with strong binding affinity were selected.
  • the MDA-MB-231 human EphA10-overexpressing cell line and the mouse EphA10-overexpressing cell line were suspended in DPBS (Corning) and 100 ⁇ l was dispensed into each e-tube so that the number of cells was 9 ⁇ 10 4 .
  • EphA10-binding humanized antibodies purified in the above Example 3 and 500 ng of mouse antibody (Chimeric #2) and IgG before humanization as a control were treated and incubated at 4°C for 30 minutes, and as a 2nd antibody, Alexa Fluor 488 anti-human IgG (Invitrogen) was treated at a ratio of 1:100, and then incubated at 4°C for 30 minutes in a light-shielded manner. After washing twice with DPBS, the mixture was analyzed using a CytoFLEX flow cytometer (Beckman Coulter).
  • the quantitative binding constants of two kinds of EphA10-specific humanized antibodies 1-1 and 1-2 were analyzed using a BLI (Bio-Layer Interferometry) equipment, Octet ® BLI system R8 (Satorius). Specifically, for immobilization, two kinds of EphA10-specific humanized antibodies (1-1 and 1-2) diluted in 200 ⁇ l of 1 ⁇ PBS at 5 ⁇ g/ml were incubated for 300 s on an Octet ® Protein A bio-sensor (Satorius).
  • EphA10-His monomer antigen protein serially diluted in 1 ⁇ PBS from 1,000 nM to 200 ⁇ l in a volume of 1 ⁇ PBS was associated with the EphA10-specific humanized antibody-coated biosensor for 300 s. Dissociation was then performed by incubating with 200 ⁇ l of 1 ⁇ PBS for 300 s, and quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius) (Fig. 7).
  • the ADC was produced by the partial reduction followed by cysteine conjugation method, which is generally used in the production of ADC. Specifically, the buffer of the EphA10-specific humanized antibodies 1-1 and 1-2 stored in 1 ⁇ PBS was exchanged with a reducing buffer (5 mM DTPA, pH 8.0 PBS) using PD MidiTrap G-25 (Cytiva, 28918008), and then concentrated to 4 mg/ml using Centrifugal filter units 10K (Merck Millipore).
  • a reducing buffer 5 mM DTPA, pH 8.0 PBS
  • the concentrated antibody was mixed with reduction buffer to obtain 2 mg/ml and Tris(2-carboxyethyl)phosphine (TCEP, sigma) 68.7 uM, and reduction reaction was performed at 37°C for 2 hours. TCEP was then removed to prevent further reduction, and exchange and concentration were performed in the same manner as the buffer exchange described above with 1 ⁇ PBS buffer (pH 7.4) using PD MidiTrap G-25 column, Centrifugal filter units 10k for conjugation.
  • TCEP Tris(2-carboxyethyl)phosphine
  • the reduced antibody at a concentration of 2 mg/ml was mixed with 63.2 uM MC-Val-Cit-PAB-MMAE (HY-15575, medchemexpress), a linker drug that reacts with cysteine, in 1 ⁇ PBS, and reacted at 4°C for 1 hour.
  • cysteine was dissolved in 1 ⁇ PBS, treated with 126.4 ⁇ M, which is twice the concentration of the treated linker-drug, and quenched at room temperature for 10 min.
  • MDA-MB-231 naive cells control group
  • mock cell line negative control group
  • hEphA10 overexpressing cell line were each dispensed at 2 ⁇ 10 4 per well in a 96-well white plate (SPL), and the following day, the ADC produced in Example 6 was diluted by concentration in RPMI (Capricorn), and 100 ⁇ l was treated to each well, followed by incubation at 37 °C for 48 hours.
  • the ADC-treated plate was incubated for 5 minutes at room temperature outside the incubator, 50 ⁇ l of CellTiter-Glo solution was added to each well, and then shaken at 500 rpm for 2 minutes. After reacting for 10 minutes on a room temperature bench in a dark place, ATP was measured using a Synergy HTX multimode reader (Biotek).
  • EphA10-targeting ADC antibody was produced by linking a low-molecular-weight substance of the payload through a linker, it was assumed that in this process, the payload was conjugated to the free cysteine of the CDR region, thereby blocking the site that binds to the antigen epitope of the EphA10-targeting humanized antibody and thereby losing antigen binding ability.
  • the binding affinity of EphA10-specific humanized antibody (1-1) and EphA10-targeting ADC antibody (1-1 (ADC)) after ADC manufacturing was analyzed by ELISA and compared.
  • the free cysteine present in the light chain of the EphA10-specific humanized antibodies 1-1 and 1-2 selected in Example 4 was selected and replaced with serine (1-1-S and 1-2-S) which has similar properties to cysteine, or alanine (1-1-A and 1-2-A) to minimize the effect of the overall structural change of the antibody, respectively, and each was cloned into a vector for light chain animal cell expression, and each antibody was expressed and purified in animal cells as in the above Example 3, and the size and purity of the purified antibodies (1-1-A, 1-1-S, 1-2-A, and 1-2-S) were analyzed through SDS-PAGE (Fig. 12).
  • Example 8 the binding affinity of antibodies (1-1-A, 1-1-S, 1-2-A, and 1-2-S) in which the free cysteine present in the light chain of two types of EphA10-specific humanized antibodies 1-1 and 1-2 was substituted with serine and alanine, respectively, to EphA10 was analyzed by ELISA.
  • an antibody library was constructed by amplifying the variable regions (VH and VL) of antibodies 1-1 and 1-2 by introducing random mutations at 0.2% through error-prone PCR.
  • the constructed DNA library was transformed into 400 ⁇ l of competent Saccharomyces cerevisiae cells by electroporation using a MicroPulser Electroporator (Bio-Rad), and then cultured in 500 ml of SD medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD Biosciences) 5.0 g/l, Na 2 HPO 4 (Junsei) 5.4 g/l, NaH 2 PO 4 .H 2 O (Samchun) 8.56 g/l, Glucose (Duksan) 20 g/l] to selectively allow the transformed yeast to survive, resulting in a yeast display library with library sizes of 7.5 ⁇ 10 7 and 1.0 ⁇ 10 8 for 1-1 and 1-2, respectively. was produced (Fig. 14).
  • human Fc-fused antigen protein EphA10-Fc was labeled with Alexa488 fluorescent molecule for screening using a flow cytometer.
  • 5.5 ⁇ 10 8 cells of the yeast library produced in Example 10 were cultured in 100 ml of SD medium lacking tryptophan at 30°C for 16 hours, and then 7 ⁇ 10 8 cells were cultured in 100 ml of SG medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD) 5.0 g/l, Na 2 HPO 4 (Junsei) 5.4 g/l, NaH 2 PO 4 .H 2 O (SAMCHUN) 8.56 g/l, Galactose (Sigma-Aldrich) 20 g/l] at 20°C for 48 hours.
  • SG medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD) 5.0 g/l, Na 2 HPO
  • the cells were washed with 1 ml of PBSB (pH 7.4), resuspended in 1 ml of PBSB (pH 7.4), and the cells showing high fluorescence signals by inducing binding to Alexa488-conjugated EphA10-Fc were separated using a flow cytometer (Bio-Rad S3
  • the recovered sub-library was collected through a sorter, Bio-Rad.
  • the recovered sub-library was subjected to a total of five rounds of selection using decreasing concentrations of Alexa488-conjugated EphA10-Fc in the same manner to enrich for cells displaying Fab variants with excellent binding affinity to EphA10 (Fig. 15).
  • Example 11 the gene sequences of Fab variant clones with excellent binding signals to EphA10 were confirmed by Sanger sequencing.
  • Example 13 In order to confirm whether the binding affinity of the 16 antibodies purified in Example 13 above increased toward EphA10, an ELISA analysis was performed. As a result, it was confirmed that the binding affinity of the humanized antibodies selected through additional engineering in Example 10 improved toward EphA10 (Fig. 18).
  • EphA10-specific humanized antibodies selected by substituting cysteines in the CDR region and undergoing further engineering two types (A.7 and 2-W) were used to produce ADCs by the cysteine conjugation method of Example 6, and analyzed by SDS-PAGE (Fig. 19).
  • the purified ADCs were analyzed by the hydrophobic interaction chromatography method (Fig. 19).
  • the produced ADCs showed peak separation by the difference in hydrophobicity that occurred according to the DAR, and the distribution and ratio of the DAR (Drug to Antibody ratio) of the ADCs were confirmed by HIC analysis.
  • the EphA10-targeting ADC (2W_ADC) produced using an antibody (2-W) in which the free cysteine present in the light chain CDR of the humanized antibody 1-2 selected in Example 4 was substituted with tryptophan
  • the EphA10-targeting ADC (A7_ADC) produced using the antibody A7 (A.7) selected through the yeast display library were analyzed by ELISA to see whether the EphA10 binding affinity of the antibodies was maintained at the same level as before the ADC.
  • Example 17 Enhanced binding affinity to EphA10 Confirmation of cancer cell killing effect of humanized antibody-based ADC
  • EphA10-His monomer antigen protein from 1,000 nM to 200 ⁇ l in 1 ⁇ PBS was bound to the biosensor coated with each EphA10-specific humanized antibody (A.7 and 2-W) for 300 s. After that, dissociation was performed by incubating with 200 ⁇ l of 1 ⁇ PBS for 300 s, and quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius).
  • an antibody binds to mouse EphA10, it can be used as a good indicator for selecting a mouse model before in vivo experiments with EphA10-targeting antibodies. Therefore, an animal cell expression vector capable of expressing the extracellular domain (ECD) of mouse EphA10 together with various fusion proteins was prepared, transfected into Expi293F cells, and cultured for 7 days. Then, centrifugation was performed and the supernatant was collected.
  • ECD extracellular domain
  • the supernatant was filtered using a 0.2 ⁇ m bottle top filter (Merck Millipore), and 1 ml of GSH resin, Protein A resin, or Ni-NTA resin was added to the filtered supernatant, and the mixture was stirred at 4 °C for 16 hours. After that, the mixture was passed through a column to recover each resin. After washing with 5 ml of 1 ⁇ PBS, the solution was eluted with 50 mM Tris-HCl (pH 8.0), 200 mM glycine (pH 2.7), or 250 mM imidazole containing 4 ml of 10 mM reduced glutathione, respectively.
  • EphA10-specific humanized antibodies A.7 and 2-W
  • ELISA was performed. Specifically, 4 ⁇ g/ml of mEphA10-Fc was diluted with 0.05 M Na 2 CO 3 (pH 9.6) solution, 50 ⁇ l each was immobilized on a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 ⁇ l of PBS containing 4% skim milk (GenomicBase) at room temperature for 2 hours.

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Abstract

The present invention relates to humanized antibodies targeting human EphA10 and use thereof. The antibody specific to EphA10 or an immunologically active fragment thereof according to the present invention specifically bind to EphA10, a highly promising target for overcoming resistance and non-responsiveness to existing immune checkpoint inhibitor therapies. When created with the antibody, an ADC was found to specifically kill cancer cells. Therefore, it can be used as a therapeutic antibody for cancer treatment leveraging cancer cell death mechanisms via immune cell activation or for treating EphA10-expressing cancers, or as a cell therapeutic for treating EphA10-expressing cancers, and can also be used for cancer diagnosis.

Description

EPHA10 표적 인간화 항체들EPHA10 targeting humanized antibodies

본 발명은 인간 EphA10 표적 인간화 항체 및 이의 용도에 관한 것이다.The present invention relates to humanized antibodies targeting human EphA10 and uses thereof.

암(cancer)은 유전자 발현의 다양한 변화에 의하여 세포의 증식과 사멸이 정상적으로 조절되지 않고 세포의 비정상적인 성장이 야기된 상태로, 인접한 조직에 침투하여 파괴하고 다른 부위로 전이하여 결국 사람이 사망에 이르게 한다. 암을 유발하는 원인, 즉 정상세포가 어떠한 기전을 거쳐 암 세포로 형질전환 되는지에 대해서는 정확히 규명되지는 않았으나, 현재까지 알려진 바에 따르면 암은 환경요인, 화학물질, 방사선, 바이러스 등 외적 요인 및 유전 인자, 면역학적 요인 등의 내적 요인 등이 복잡하게 얽혀 결과적으로 암을 유발하는 것으로 알려져 있다. 암은 혈액 세포 수에 이상을 보이는 혈액암과 신체 내에서 일정한 경도와 형태를 갖는 세포 덩어리 형태의 고형암으로 크게 분류된다. 암은 혈액, 조직 신체의 거의 전 부위에서 발생할 수 있으며, 폐암, 위암, 유방암, 구강암, 간암, 자궁암, 식도암, 피부암 등을 예로 들 수 있다. 암의 치료에는 대부분 수술이나 방사선 요법 및 세포 증식을 억제하는 화학 요법제를 이용한 항암제 치료가 주된 방법이다. 암 치료를 위한 의약품은 크게 저분자 의약품과 고분자 의약품으로 나뉘며 특이성이 없어 부작용이 상대적으로 큰 저분자 의약품에 비해 특이성이 있는 고분자 의약품이 치료제로서 각광을 받고 있다. Cancer is a condition in which cell proliferation and death are not normally controlled due to various changes in gene expression, resulting in abnormal cell growth, which infiltrates and destroys adjacent tissues and metastasizes to other parts, ultimately leading to death. The exact cause of cancer, i.e. the mechanism through which normal cells are transformed into cancer cells, has not been identified, but according to what is currently known, cancer is known to be caused by a complex intertwining of external factors such as environmental factors, chemicals, radiation, and viruses, and internal factors such as genetic factors and immunological factors. Cancer is broadly classified into blood cancer, which shows abnormalities in the number of blood cells, and solid cancer, which is a lump of cells with a certain hardness and shape in the body. Cancer can occur in almost any part of the blood, tissue, and body, and examples include lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. The main methods of cancer treatment are surgery, radiation therapy, and anticancer treatment using chemotherapy agents that suppress cell proliferation. Medicines for cancer treatment are largely divided into small molecule drugs and large molecule drugs. Compared to small molecule drugs, which have relatively more side effects due to lack of specificity, large molecule drugs with higher specificity are receiving attention as treatment agents.

단백질 치료제들은 질병 타깃들에 아주 높은 특이성과 낮은 부작용과 독성을 보이기 때문에 비특이적인 저분자 화합물 치료제들을 아주 빠른 속도로 대체하여 임상에서 널리 이용되고 있으며, 현재 임상에 이용되고 있는 단백질 치료제들 중 항체 치료제들과 항체 Fc영역을 융합된 Fc-융합 단백질 치료제들이 주종을 이루고 있다. 치료용 항체는 기존의 저분자 약물에 비해 타깃에 매우 높은 특이성을 보이며, 생체 독성이 낮고 부작용이 적을 뿐만 아니라, 약 3주의 우수한 혈중 반감기를 가지기 때문에 가장 효과적인 암 치료방법 중의 하나로 여겨지고 있다. 실제로 전 세계의 거대 제약회사들과 연구소들에서 암 발병 원인인자를 비롯한 암세포에 특이적으로 결합하여 효과적으로 제거하는 치료용 항체의 연구 개발에 박차를 가하고 있다. 치료용 항체 의약품 개발 기업으로는 로슈, 암젠, 존슨앤존슨, 애보트, 비엠에스 등의 제약 기업이 주를 이루고 있으며, 특히 로슈는 항암 치료 목적의 허셉틴 (Herceptin), 아바스틴 (Avastin), 리툭산 (Rituxan) 등이 대표적 상품으로 이 세 가지 치료용 항체로 2012년 세계시장에서 약 195억 달러의 매출을 달성하는 등 큰 이윤을 창출하고 있을 뿐 아니라, 세계의 항체 의약품 시장을 이끌고 있다. 레미케이드(Remicade)를 개발한 존슨앤존슨 역시 매출의 증가로 세계 항체 시장에서 빠르게 성장해나가고 있으며, 애보트와 비엠에스 등의 제약 기업 역시 개발 막바지 단계의 치료용 항체를 다수 보유하고 있는 것으로 알려져 있다. 이에 따른 결과로 저분자 의약품이 주도권을 가지고 있던 세계 제약 시장에서 질병 타깃에 특이적이고 부작용이 낮은 치료용 항체를 포함한 바이오 의약품이 빠르게 그 자리를 대체해 나가고 있다. Because protein therapeutics show very high specificity for disease targets and low side effects and toxicity, they are rapidly replacing non-specific small molecule compound therapeutics and are widely used in clinical settings. Among the protein therapeutics currently used in clinical settings, antibody therapeutics and Fc-fusion protein therapeutics that are fused with antibody Fc regions are the main types. Therapeutic antibodies show very high specificity for their targets compared to existing small molecule drugs, and are considered one of the most effective cancer treatments because they not only have low biotoxicity and few side effects, but also have an excellent blood half-life of about 3 weeks. In fact, large pharmaceutical companies and research institutes around the world are spurring research and development of therapeutic antibodies that specifically bind to cancer cells, including carcinogenic factors, and effectively remove them. Therapeutic antibody drug development companies include pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS. In particular, Roche is leading the global antibody drug market, generating huge profits with its representative products such as Herceptin, Avastin, and Rituxan for anticancer treatment, achieving sales of approximately 19.5 billion dollars in the global market in 2012 with these three therapeutic antibodies. Johnson & Johnson, which developed Remicade, is also rapidly growing in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have many therapeutic antibodies in the final stages of development. As a result, in the global pharmaceutical market where small molecule drugs were dominant, biopharmaceuticals including therapeutic antibodies that are specific to disease targets and have low side effects are quickly taking their place.

암세포들은 면역세포들에 의한 살상 작용기작을 회피하기 위해 정상세포들이 면역세포 활성화를 억제할 때 이용되는 면역관문(immune checkpoint) 단백질을 세포 표면에 발현하고 있어, 최근 암을 치료하기 위한 방법으로써 면역관문 억제제(immune checkpoint inhibitor)에 대한 연구가 활발히 진행되고 있다 (Immunity. 2018 Mar 20;48(3):434-452.). 면역관문 억제제는 T 세포 억제에 관여하는 면역관문 단백질(immune checkpoint protein)의 활성을 차단하여 T 세포를 활성화시킴으로써 암세포를 공격하는 약제로서 대표적으로 CTLA 4, PD-1, PD-L1 등을 인식하는 항체를 사용한다. 그러나, 면역관문 억제제(immune checkpoint inhibitor), 면역세포치료제 (immune cell therapy), 치료용 항체(therapeutic antibody) 및 항암 백신 (anticancer vaccine)을 포함하는 면역 항암제에 대한 암 환자의 반응률은 아직까지 15 내지 45% 수준에 머무르고 있고, 대부분의 고형암종은 면역항암제에 불응성과 내성을 획득하는 문제가 있다. 이에, anti-PD-1 항체 치료제를 반복적으로 적용함으로써 anti-PD-1 치료제에 저항성을 획득하도록 유도한 마우스 모델과 인간 암세포주 등을 활용한 종래의 연구들에서 면역관문 억제인자를 표적하는 치료제에 불응성을 유발하는 인자로서 세포막 단백질인 EphA10이 동정되었다. EphA10은 RTKs(receptor tyrosine kinases)의 가장 큰 패밀리인 Ephrin 수용체의 한 종류로써 다른 Ephrin 수용체들과 달리 EphA10은 그 기능 연구가 거의 이루어지지 않은 매우 신규한 수용체이며, 적절한 치료제가 없고 예후가 매우 좋지 않은 유방암인 TNBC (Triple Negative Breast Cancer)을 포함한 모든 유방암에서 발현량이 크게 증가하며, 폐암, 췌장암, 전립선암, 위암, 갑상선암 등에서도 정상조직에 비해 유의하게 발현량이 증가되는 것으로 보고되었다.Cancer cells express immune checkpoint proteins on their cell surface, which are used by normal cells to suppress immune cell activation in order to avoid the killing mechanism by immune cells. Recently, research on immune checkpoint inhibitors as a method of treating cancer has been actively conducted (Immunity. 2018 Mar 20;48(3):434-452.). Immune checkpoint inhibitors are drugs that attack cancer cells by blocking the activity of immune checkpoint proteins involved in T cell suppression and activating T cells. Representative examples use antibodies that recognize CTLA 4, PD-1, and PD-L1. However, the response rate of cancer patients to immunotherapy, including immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies, and anticancer vaccines, still remains at the level of 15 to 45%, and most solid tumors have the problem of acquiring nonresponsiveness and resistance to immunotherapy. Therefore, in previous studies utilizing mouse models and human cancer cell lines that were induced to acquire resistance to anti-PD-1 therapy by repeated application of anti-PD-1 antibody therapy, the cell membrane protein EphA10 was identified as a factor that causes nonresponsiveness to therapeutics targeting immune checkpoint inhibitors. EphA10 is a type of Ephrin receptor, the largest family of RTKs (receptor tyrosine kinases). Unlike other Ephrin receptors, EphA10 is a very novel receptor whose function has been rarely studied. Its expression level is significantly increased in all breast cancers, including TNBC (Triple Negative Breast Cancer), a type of breast cancer with no appropriate treatment and a very poor prognosis, and it has also been reported that its expression level is significantly increased in lung cancer, pancreatic cancer, prostate cancer, stomach cancer, and thyroid cancer compared to normal tissues.

본 발명의 목적은 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제공하는 것이다.The purpose of the present invention is to provide an antibody specific for EphA10 or a fragment thereof having immunological activity.

또한, 본 발명의 목적은 EphA10에 특이적인 항체-약물 접합체를 제공하는 것이다.Furthermore, it is an object of the present invention to provide an antibody-drug conjugate specific for EphA10.

또한, 본 발명의 목적은 이중특이적 또는 다중특이적 항체를 제공하는 것이다.It is also an object of the present invention to provide bispecific or multispecific antibodies.

또한, 본 발명의 목적은 키메라 항원 수용체(Chimeric antigen receptor: CAR)를 제공하는 것이다.Furthermore, it is an object of the present invention to provide a chimeric antigen receptor (CAR).

또한, 본 발명의 목적은 키메라 항원 수용체 발현 세포를 제공하는 것이다.Furthermore, it is an object of the present invention to provide a chimeric antigen receptor expressing cell.

또한, 본 발명의 목적은 T 세포 관여자(T-cell engager)를 제공하는 것이다.Furthermore, it is an object of the present invention to provide a T-cell engager.

또한, 본 발명의 목적은 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.

또한, 본 발명의 목적은 암 진단용 조성물을 제공하는 것이다.In addition, it is an object of the present invention to provide a composition for cancer diagnosis.

또한, 본 발명의 목적은 암의 예방 또는 치료 용도를 제공하는 것이다.In addition, it is an object of the present invention to provide a use for preventing or treating cancer.

또한, 본 발명의 목적은 암의 치료 방법을 제공하는 것이다.Furthermore, it is an object of the present invention to provide a method for treating cancer.

아울러, 본 발명의 목적은 암 진단 용도를 제공하는 것이다.In addition, it is an object of the present invention to provide a cancer diagnosis application.

상기 과제를 해결하기 위하여, 본 발명은 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편을 제공한다.To solve the above problem, the present invention provides an antibody specific for EphA10 or a fragment thereof having immunological activity.

또한, 본 발명은 상기 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 EphA10에 특이적인 항체-약물 접합체를 제공한다.Additionally, the present invention provides an antibody-drug conjugate specific for EphA10 comprising the antibody or a fragment thereof having immunological activity.

또한, 본 발명은 상기 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 이중특이적 또는 다중특이적 항체를 제공한다.The present invention also provides a bispecific or multispecific antibody comprising the antibody or a fragment having immunological activity thereof.

또한, 본 발명은 상기 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 키메라 항원 수용체를 제공한다.The present invention also provides a chimeric antigen receptor comprising the antibody or a fragment thereof having immunological activity.

또한, 본 발명은 키메라 항원 수용체 발현 세포를 제공한다.Additionally, the present invention provides a chimeric antigen receptor expressing cell.

또한, 본 발명은 상기 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 T 세포 관여자를 제공한다.Additionally, the present invention provides a T cell engager comprising the antibody or a fragment thereof having immunological activity.

또한, 본 발명은 암의 예방 또는 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing or treating cancer.

또한, 본 발명은 암 진단용 조성물을 제공한다.In addition, the present invention provides a composition for cancer diagnosis.

또한, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자의 암의 예방 또는 치료 용도를 제공한다.The present invention also provides a use of the antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager for the prevention or treatment of cancer.

또한, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자를 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암의 치료 방법을 제공한다.The present invention also provides a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager.

아울러, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자의 암 진단 용도를 제공한다.In addition, the present invention provides a use of the antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager for the diagnosis of cancer.

본 발명의 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편은 기존 면역관문 억제인자 표적 치료제의 불응성과 내성 극복을 위해 매우 유력한 타겟인 EphA10에 특이적으로 결합하며, 이를 이용하여 ADC를 제작한 경우 암세포를 특이적으로 사멸시키는 것으로 나타났으므로, 면역세포 활성화에 의한 암세포 사멸 기작을 이용하는 암 치료용 항체 치료제, EphA10 발현 암 치료를 위한 항체 치료제 또는 EphA10 발현 암 치료를 위한 세포 치료제로 이용할 수 있는 효과가 있고, 암의 진단에도 이용할 수 있는 효과가 있다.The antibody specific for EphA10 of the present invention or a fragment thereof having immunological activity specifically binds to EphA10, which is a very potent target for overcoming the non-responsiveness and resistance of existing immune checkpoint inhibitor targeted therapeutics, and when an ADC is produced using the antibody, it was shown to specifically kill cancer cells. Therefore, it can be used as an antibody therapeutic for treating cancer utilizing a cancer cell death mechanism by immune cell activation, an antibody therapeutic for treating EphA10-expressing cancer, or a cell therapeutic for treating EphA10-expressing cancer, and it also has an effect that can be used for the diagnosis of cancer.

도 1은 EphA10 10종 항원 단백질의 동물세포에서 발현 및 정제한 후 SDS-PAGE 젤로 크기와 순도를 분석한 도이다. Figure 1 is a diagram showing the size and purity of 10 EphA10 antigen proteins expressed and purified in animal cells using SDS-PAGE gel.

도 2는 EphA10 표적 마우스 항체의 아미노산 서열을 분석한 결과 및 이를 바탕으로 제작한 chimeric #2 항체의 EphA10에 대한 결합력을 분석한 결과이다. Figure 2 shows the results of analyzing the amino acid sequence of the EphA10 target mouse antibody and the results of analyzing the binding ability of the chimeric #2 antibody produced based on the amino acid sequence to EphA10.

도 3은 EphA10 표적 인간화 항체의 아미노산 서열 및 항체 정보를 나타낸 도이다. Figure 3 is a diagram showing the amino acid sequence and antibody information of an EphA10 targeting humanized antibody.

도 4는 EphA10 표적 인간화 항체들을 동물세포에서 발현 및 정제한 후 이의 발현량을 분석한 결과와 정제한 인간화 항체들의 크기와 순도를 SDS-PAGE 젤로 확인한 도이다.Figure 4 is a diagram showing the results of analyzing the expression levels of EphA10 targeting humanized antibodies expressed and purified in animal cells and confirming the size and purity of the purified humanized antibodies using an SDS-PAGE gel.

도 5는 EphA10 표적 인간화 항체 5종의 EphA10에 대한 결합력을 ELISA로 분석한 결과이다. Figure 5 shows the results of ELISA analysis of the binding affinity of five EphA10-targeting humanized antibodies to EphA10.

도 6은 EphA10 표적 인간화 항체 5종의 인간 및 마우스 세포에서 발현되는 EphA10 대한 결합력을 확인한 도이다.Figure 6 is a diagram confirming the binding affinity of five types of EphA10-targeting humanized antibodies to EphA10 expressed in human and mouse cells.

도 7은 EphA10 표적 인간화 항체들의 EphA10에 대한 정량적인 결합 상수 분석 결과를 나타낸 도이다. Figure 7 shows the binding of EphA10 targeting humanized antibodies to EphA10. This diagram shows the results of quantitative binding constant analysis.

도 8은 결합력이 높아 선발된 EphA10 특이적 항체 (1-1 및 1-2)로 제조한 ADC의 항체-약물 결합을 분석한 결과이다.Figure 8 shows the results of analyzing the antibody-drug binding of ADCs manufactured with EphA10-specific antibodies (1-1 and 1-2) selected for their high binding affinity.

도 9는 EphA10 특이적 항체 (1-1 및 1-2)로 제작된 ADC의 암세포 사멸 효능을 확인한 도이다.Figure 9 is a diagram confirming the cancer cell killing efficacy of ADC produced with EphA10 specific antibodies (1-1 and 1-2).

도 10은 EphA10 특이적 인간화 항체의 서열을 분석하여 경쇄에 존재하는 자유 시스테인 잔기를 확인한 도이다. Figure 10 is a diagram showing the sequence of an EphA10-specific humanized antibody, identifying free cysteine residues present in the light chain.

도 11은 EphA10 특이적 인간화 항체 (1-1) 및 이를 이용하여 제작한 EphA10 표적 ADC 항체 (1-1 (ADC))의 EphA10에 대한 결합력을 ELISA로 분석 결과를 나타낸 도이다:Figure 11 is a diagram showing the results of ELISA analysis of the binding affinity of EphA10-specific humanized antibody (1-1) and EphA10-targeting ADC antibody (1-1 (ADC)) produced using the same to EphA10:

1-1: EphA10 특이적 인간화 항체; 및1-1: EphA10 specific humanized antibody; and

1-1 (ADC): EphA10 표적 ADC 항체.1-1 (ADC): EphA10 targeting ADC antibody.

도 12는 EphA10 특이적 항체 1-1 및 1-2의 경쇄에 존재하는 자유 시스테인 잔기를 각각 알라닌(alanine, A) 또는 세린(serine, S)으로 치환한 ADC 효능 개선된 EphA10 특이적 인간화 항체들을 동물세포에서 발현 및 정제한 후 이의 크기와 순도를 SDS-PAGE 젤로 확인한 도이다.Figure 12 is a diagram showing the size and purity of EphA10-specific humanized antibodies with improved ADC efficacy, in which the free cysteine residues present in the light chains of EphA10-specific antibodies 1-1 and 1-2 were substituted with alanine (A) or serine (S), respectively, expressed and purified in animal cells, and confirmed by SDS-PAGE gel.

도 13은 ADC 효능 개선된 EphA10 특이적 인간화 항체들의 EphA10에 대한 결합력을 분석한 결과이다.Figure 13 shows the results of analyzing the binding affinity of EphA10-specific humanized antibodies with improved ADC efficacy to EphA10.

도 14는 EphA10 표적 인간화 항체들의 결합력 증가를 위한 효모 라이브러리 구축 및 스크리닝 전략을 나타낸 도이다. Figure 14 is a diagram illustrating a yeast library construction and screening strategy for increasing the binding affinity of humanized antibodies targeting EphA10.

도 15는 EphA10 표적 인간화 항체들의 결합력 증가를 위해 유세포 분리기를 이용한 반복적인 스크리닝 과정을 나타낸 도이다. Figure 15 is a diagram showing an iterative screening process using a flow cytometer to increase the binding affinity of humanized antibodies targeting EphA10.

도 16은 유세포 분리기를 이용한 탐색을 통해 발굴된 10종의 항체 (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N 및 2-W) 및 추가로 자유 시스테인을 치환한 6종 (1-R, 1-W, 1-Y, 2-N, 2-R 및 2-Y)의 인간화 항체의 아미노산 서열 분석결과이다. Figure 16 shows the amino acid sequence analysis results of 10 antibodies (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) discovered through exploration using a flow cytometer and 6 additional humanized antibodies (1-R, 1-W, 1-Y, 2-N, 2-R, and 2-Y) with free cysteines substituted.

도 17은 EphA10에 대한 결합력이 증가한 인간화 항체 16종을 동물세포에서 발현 및 정제한 후 이의 크기와 순도를 SDS-PAGE 젤로 확인한 도이다.Figure 17 is a diagram showing the size and purity of 16 humanized antibodies with increased binding affinity to EphA10 expressed and purified in animal cells, confirmed by SDS-PAGE gel.

도 18은 EphA10에 대한 결합력이 증가한 인간화 항체 16종의 EphA10에 대한 결합력을 분석한 결과이다.Figure 18 shows the results of analyzing the binding affinity to EphA10 of 16 humanized antibodies with increased binding affinity to EphA10.

도 19는 EphA10에 대한 결합력이 증가한 인간화 항체 16종 중 2종 (A7 및 2W)으로 제작한 ADC의 항체-약물 결합을 분석한 결과이다.Figure 19 shows the results of analyzing the antibody-drug binding of ADCs produced with 2 types (A7 and 2W) among 16 humanized antibodies with increased binding affinity to EphA10.

도 20은 EphA10 특이적 항체 1-1과 이로 제조한 ADC (1-1_ADC), EphA10에 대한 결합력이 증가한 인간화 항체 A7 및 2W와 이를 이용하여 제작한 EphA10 표적 ADC 항체 (A7_ADC 및 2W_ADC)의 EphA10에 대한 결합력을 ELISA로 분석한 결과를 나타낸 도이다.Figure 20 is a diagram showing the results of ELISA analysis of the binding affinity to EphA10 of EphA10-specific antibody 1-1 and ADC manufactured using the same (1-1_ADC), humanized antibodies A7 and 2W with increased binding affinity to EphA10, and EphA10-targeting ADC antibodies manufactured using the same (A7_ADC and 2W_ADC).

도 21은 A7_ADC 및 2W_ADC의 암세포 사멸 효능을 확인한 도이다.Figure 21 is a diagram confirming the cancer cell killing efficacy of A7_ADC and 2W_ADC.

도 22는 EphA10에 대한 결합력이 증가한 인간화 항체 A7 및 2W의 EphA10에 대한 정량적인 결합 상수 분석 결과를 나타낸 도이다. Figure 22 is a diagram showing the results of quantitative binding constant analysis of humanized antibodies A7 and 2W with increased binding affinity to EphA10 for EphA10.

도 23은 4종의 마우스 EphA10 항원을 포함하는 융합 단백질을 동물세포에서 발현 및 정제한 후 이의 크기와 순도를 SDS-PAGE 젤로 확인하고, 이의 활성을 ELISA로 분석한 도이다.Figure 23 shows the size and purity of a fusion protein containing four mouse EphA10 antigens expressed and purified in animal cells, confirmed by SDS-PAGE gel, and its activity analyzed by ELISA.

도 24는 EphA10에 대한 결합력이 증가한 인간화 항체 A7 및 2W의 마우스 EphA10에 대한 결합력을 ELISA로 분석한 결과를 나타낸 도이다.Figure 24 is a diagram showing the results of ELISA analysis of the binding affinity of humanized antibodies A7 and 2W with increased binding affinity to EphA10 to mouse EphA10.

이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail with reference to the attached drawings as embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terminology used in this specification is a term used to appropriately express the preferred embodiment of the present invention, and this may vary depending on the intention of the user or operator, or the custom of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components can be included, unless specifically stated otherwise.

본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 통합된다.All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications mentioned as references in this specification are incorporated into the present invention.

본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대한 통상의 1문자 및 3문자 코드가 사용될 뿐만 아니라 Aib(α-아미노이소부티르산), Sar(N-methylglycine) 등과 같은 다른 아미노산에 대해 일반적으로 허용되는 3문자 코드가 사용된다. 또한 본 발명에서 약어로 언급된 아미노산은 하기와 같이 IUPAC-IUB 명명법에 따라 기재되었다:Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. In addition, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

알라닌: A, 아르기닌: R, 아스파라긴: N, 아스파르트산: D, 시스테인: C, 글루탐산: E, 글루타민: Q, 글리신: G, 히스티딘: H, 이소류신: I, 류신: L, 리신: K, 메티오닌: M, 페닐알라닌: F, 프롤린: P, 세린: S, 트레오닌: T, 트립토판: W, 티로신: Y 및 발린: V. Alanine: A, arginine: R, asparagine: N, aspartic acid: D, cysteine: C, glutamic acid: E, glutamine: Q, glycine: G, histidine: H, isoleucine: I, leucine: L, lysine: K, methionine: M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

일 측면에서, 본 발명은 EphA10(Eph receptor A10)에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편에 관한 것이다.In one aspect, the present invention relates to an antibody specific for EphA10 (Eph receptor A10) or a fragment thereof having immunological activity.

일 구현예에서, EphA10는 인간 EphA10 또는 마우스 EphA10일 수 있다.In one embodiment, EphA10 can be human EphA10 or mouse EphA10.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 EphA10의 세포 외부 영역(Extra Cellular Domain, ECD) 또는 세포 외부 영역의 FNIII 도메인에 특이적으로 결합할 수 있다.In one embodiment, the antibody or immunologically active fragment thereof of the present invention can specifically bind to the extra cellular domain (ECD) of EphA10 or the FNIII domain of the extra cellular domain.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 서열번호 1 내지 3의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRH(Complementarity determining regions Heavy chain)1, 서열번호 4 내지 6의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRH2, 및 서열번호 7 또는 8의 아미노산 서열을 포함하는 CDRH3를 포함하는 VH 도메인을 포함할 수 있다.In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 3, CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 4 to 6, and CDRH3 comprising an amino acid sequence of SEQ ID NOs: 7 or 8.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 서열번호 9 내지 14의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRL1, 서열번호 15 또는 16의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 17 내지 19의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRL3를 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, the antibody of the present invention or an immunologically active fragment thereof can comprise a VL domain comprising CDRL1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 9 to 14, CDRL2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 15 or 16, and CDRL3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 서열번호 20 내지 25의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 VH 도메인을 포함할 수 있다.In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a VH domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 20 to 25.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 서열번호 26 내지 45의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a VL domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 26 to 45.

일 구현예에서, 본 발명의 항체 1-1는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 26의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

일 구현예에서, 본 발명의 항체 1-2는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 27의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-2 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 27.

일 구현예에서, 본 발명의 항체 A.3는 서열번호 21의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 28의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody A.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 21 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.

일 구현예에서, 본 발명의 항체 A.7는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 29의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody A.7 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 29.

일 구현예에서, 본 발명의 항체 A.8는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 30의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody A.8 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 30.

일 구현예에서, 본 발명의 항체 B.1는 서열번호 22의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 31의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody B.1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 22 and a VL domain comprising the amino acid sequence of SEQ ID NO: 31.

일 구현예에서, 본 발명의 항체 B.3는 서열번호 23의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 32의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody B.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 32.

일 구현예에서, 본 발명의 항체 B.7는 서열번호 24의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 33의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody B.7 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 24 and a VL domain comprising the amino acid sequence of SEQ ID NO: 33.

일 구현예에서, 본 발명의 항체 2.1는 서열번호 25의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 34의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2.1 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34.

일 구현예에서, 본 발명의 항체 2.3는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 35의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2.3 of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 35.

일 구현예에서, 본 발명의 항체 1-A는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 36의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-A of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

일 구현예에서, 본 발명의 항체 1-N는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 37의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-N of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 37.

일 구현예에서, 본 발명의 항체 1-R는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 38의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-R of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.

일 구현예에서, 본 발명의 항체 1-W는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 39의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-W of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 39.

일 구현예에서, 본 발명의 항체 1-Y는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 40의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 1-Y of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 40.

일 구현예에서, 본 발명의 항체 2-A는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 41의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2-A of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 41.

일 구현예에서, 본 발명의 항체 2-W는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 42의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2-W of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 42.

일 구현예에서, 본 발명의 항체 2-N는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 43의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2-N of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 43.

일 구현예에서, 본 발명의 항체 2-R는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 44의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2-R of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44.

일 구현예에서, 본 발명의 항체 2-Y는 서열번호 20의 아미노산 서열을 포함하는 VH 도메인 및 서열번호 45의 아미노산 서열을 포함하는 VL 도메인을 포함할 수 있다.In one embodiment, antibody 2-Y of the present invention may comprise a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and a VL domain comprising the amino acid sequence of SEQ ID NO: 45.

일 구현예에서, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 인간 기원의 중쇄 또는 경쇄 가변 영역, 중쇄 또는 경쇄 불변 영역, 프레임워크 영역, 또는 이의 임의의 부분을 함께 포함할 수 있다.In one embodiment, the antibody or immunologically active fragment thereof of the present invention may comprise a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof of human origin.

일 구현예에서, 면역학적 활성을 가진 단편은 Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv(single chain fragment variable), Fv, 단일쇄 항체, Fv 이량체, 상보성 결정 영역 단편, 인간화 항체, 키메라 항체 및 디아바디(diabody)로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며, 인간화 항체, Fab 또는 scFv인 것이 더욱 바람직하다.In one embodiment, the fragment having immunological activity can be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody, more preferably a humanized antibody, Fab or scFv.

일 구현예에서, 본 발명의 항체는 단일클론항체일 수 있다.In one embodiment, the antibody of the invention may be a monoclonal antibody.

상기 항체는 전체(whole) 항체 형태일 뿐 아니라 항체 분자의 기능적인 단편을 포함한다. 전체 항체는 2개의 전체 길이의 경쇄(light chain) 및 2개의 전체 길이의 중쇄(heavy chain)를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합(disulfide bond)으로 연결되어 있다. 항체 분자의 기능적인 단편이란 항원 결합 기능을 보유하고 있는 단편을 뜻하며, 항체 단편의 예는 (i) 경쇄의 가변영역(VL) 및 중쇄의 가변영역(VH)과 경쇄의 불변영역(CL) 및 중쇄의 첫번째 불변 영역(CH1)으로 이루어진 Fab 단편; (ii) VH 및 CH1 도메인으로 이루어진 Fd 단편; (iii) 단일 항체의 VL 및 VH 도메인으로 이루어진 Fv 단편; (iv) VH 도메인으로 이루어진 dAb 단편(Ward ES et al., Nature 341:544-546 (1989)]; (v) 분리된 CDR 영역; (vi) 2개의 연결된 Fab 단편을 포함하는 2가 단편인 F(ab')2 단편; (vii) VH 도메인 및 VL 도메인이 항원 결합 부위를 형성하도록 결합시키는 펩타이드 링커에 의해 결합된 단일쇄 Fv 분자(scFv); (viii) 이특이적인 단일쇄 Fv 이량체(PCT/US92/09965) 및 (ix) 유전자 융합에 의해 제작된 다가 또는 다특이적인 단편인 디아바디(diabody) WO94/13804) 등을 포함한다. The above antibody is in the form of a whole antibody as well as a functional fragment of an antibody molecule. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment having an antigen-binding function, and examples of antibody fragments include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a first constant region (CH1) of a heavy chain; (ii) a Fd fragment consisting of a VH and a CH1 domain; (iii) an Fv fragment consisting of a VL and a VH domain of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) isolated CDR regions; (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer (PCT/US92/09965) and (ix) a diabody, a multivalent or multispecific fragment produced by gene fusion (WO94/13804).

본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 동물 유래 항체, 키메릭 항체, 인간화 항체, 인간 항체, 및 이들의 면역학적 활성을 가진 단편으로 이루어진 군에서 선택된 것일 수 있다. 상기 항체는 재조합적 또는 합성적으로 생산된 것일 수 있다.The antibody of the present invention or a fragment thereof having immunological activity may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof having immunological activity. The antibody may be produced recombinantly or synthetically.

원하는 항원을 피면역 동물에게 면역시켜 생산하는 동물 유래 항체는 일반적으로 치료 목적으로 인간에 투여시 면역거부반응이 일어날 수 있으며, 이러한 면역거부반응을 억제하고자 키메릭 항체(chimeric antibody)가 개발되었다. 키메릭 항체는 유전공학적 방법을 이용하여 항-아이소타입(anti-isotype) 반응의 원인이 되는 동물 유래 항체의 불변 영역을 인간 항체의 불변 영역으로 치환한 것이다. 키메릭 항체는 동물 유래 항체에 비하여 항-아이소타입 반응에 있어서 상당 부분 개선되었으나, 여전히 동물 유래 아미노산들이 가변 영역에 존재하고 있어 잠재적인 항-이디오타입(anti-idiotypic) 반응에 대한 부작용을 내포하고 있다. 이러한 부작용을 개선하고자 개발된 것이 인간화 항체(humanized antibody)이다. 이는 키메릭 항체의 가변 영역 중 항원의 결합에 중요한 역할을 하는 CDR(complementaritiy determining regions) 부위를 인간 항체 골격(framework)에 이식하여 제작된다.Animal-derived antibodies produced by immunizing a desired antigen to an immunized animal can generally cause an immune rejection reaction when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection reactions. Chimeric antibodies are produced by replacing the constant region of an animal-derived antibody, which causes an anti-isotype reaction, with a constant region of a human antibody using genetic engineering methods. Although chimeric antibodies have significantly improved the anti-isotype reaction compared to animal-derived antibodies, they still contain animal-derived amino acids in the variable region, which carries the side effect of a potential anti-idiotypic reaction. Humanized antibodies have been developed to improve such side effects. These are produced by grafting the CDR (complementarity determining regions), which play an important role in antigen binding among the variable regions of chimeric antibodies, onto a human antibody framework.

인간화 항체를 제작하기 위한 CDR 이식(grafting) 기술에 있어서 가장 중요한 것은 동물 유래 항체의 CDR 부위를 가장 잘 받아들일 수 있는 최적화된 인간 항체를 선정하는 것이며, 이를 위하여 항체 데이터베이스의 활용, 결정구조(crystal structure)의 분석, 분자모델링 기술 등이 활용된다. 그러나, 최적화된 인간 항체 골격에 동물 유래 항체의 CDR 부위를 이식할지라도 동물 유래 항체의 골격에 위치하면서 항원 결합에 영향을 미치는 아미노산이 존재하는 경우가 있기 때문에, 항원 결합력이 보존되지 못하는 경우가 상당수 존재하므로, 항원 결합력을 복원하기 위한 추가적인 항체 공학 기술의 적용은 필수적이라고 할 수 있다.The most important thing in the CDR grafting technology for producing humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. For this purpose, antibody databases, crystal structure analysis, and molecular modeling technology are utilized. However, even when the CDR region of an animal-derived antibody is grafted onto an optimized human antibody framework, there are many cases where the antigen binding ability is not preserved because there are amino acids that affect antigen binding while located in the framework of the animal-derived antibody. Therefore, the application of additional antibody engineering technology to restore antigen binding ability can be said to be essential.

상기 항체 또는 이의 면역학적 활성을 가진 단편은 생체에서 분리된 (생체에 존재하지 않는) 것 또는 비자연적으로 생산(non-naturally occurring)된 것일 수 있으며, 예컨대, 합성적 또는 재조합적으로 생산된 것일 수 있다.The above antibody or fragment having immunological activity may be isolated from a living organism (not existing in the organism) or non-naturally occurring, for example, may be synthetically or recombinantly produced.

본 발명에서 "항체"라 함은, 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서, 그 종류는 특별히 제한되지 않으며, 자연적 또는 비자연적(예컨대, 합성적 또는 재조합적)으로 얻어질 수 있다. 항체는 생체 외뿐 아니라 생체 내에서도 매우 안정하고 반감기가 길기 때문에 대량 발현 및 생산에 유리하다. 또한, 항체는 본질적으로 다이머(dimer) 구조를 가지므로 접착능(avidity)이 매우 높다. 완전한 항체는 2개의 전장(full length) 경쇄 및 2개의 전장 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 이황화 결합으로 연결되어 있다. 항체의 불변 영역은 중쇄 불변 영역과 경쇄 불변 영역으로 나뉘어지며, 중쇄 불변 영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고, 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변 영역은 카파(κ) 및 람다(λ) 타입을 가진다.In the present invention, the term "antibody" means a substance produced by antigen stimulation in the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are very stable not only in vitro but also in vivo and have a long half-life, so they are advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, so they have very high avidity. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has the kappa (κ) and lambda (λ) types.

본 발명에서 용어, "중쇄(heavy chain)"는 항원에 특이성을 부여하기 위해 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VH 및 3개의 불변 영역 도메인 CH1 , CH2 및 CH3 과 힌지(hinge)를 포함하는 전장 중쇄 및 이의 단편을 모두 포함하는 의미로 해석된다. 또한, 용어 "경쇄(light chain)"는 항원에 특이성을 부여하기 위한 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VL 및 불변 영역 도메인 CL을 포함하는 전장 경쇄 및 이의 단편을 모두 포함하는 의미로 해석된다.In the present invention, the term "heavy chain" is interpreted to mean a full-length heavy chain and fragments thereof, including a variable region domain V H including an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains C H1 , C H2 and C H3 and a hinge. In addition, the term "light chain" is interpreted to mean a full-length light chain and fragments thereof, including a variable region domain V L including an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain C L .

본 발명에서 용어, "가변 영역(variable region) 또는 가변 부위 (variable domain)"는 항원과 특이적으로 결합하는 기능을 수행하면서 서열상의 많은 변이를 보이는 항체 분자의 부분을 의미하고, 가변 영역에는 상보성 결정 영역인 CDR1, CDR2 및 CDR3가 존재한다. 상기 CDR 사이에는 프레임 워크 영역(framework region, FR) 부분이 존재하여 CDR 고리를 지지해주는 역할을 한다. 상기 "상보성 결정 영역"은 항원의 인식에 관여하는 고리모양의 부위로서 이 부위의 서열이 변함에 따라 항체의 항원에 대한 특이성이 결정된다.In the present invention, the term "variable region or variable domain" means a portion of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen, and the variable region includes complementarity determining regions, CDR1, CDR2, and CDR3. Between the CDRs, a framework region (FR) portion exists that supports the CDR ring. The "complementarity determining region" is a ring-shaped portion involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this portion changes.

본 발명에서 용어, "scFv(single chain fragment variable)"는 유전자 재조합을 통해 항체의 가변영역만을 발현시켜 만든 단쇄항체를 말하며, 항체의 VH 영역과 VL 영역을 짧은 펩티드 사슬로 연결한 단일쇄 형태의 항체를 말한다. 상기 용어 "scFv"는 달리 명시되지 않거나, 문맥상 달리 이해되는 것이 아니라면, 항원 결합 단편을 비롯한 scFv 단편을 포함하고자 한다. 이는 통상의 기술자에게 자명한 것이다.In the present invention, the term "scFv (single chain fragment variable)" refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in the form of a single chain in which the VH region and the VL region of an antibody are connected by a short peptide chain. The term "scFv" is intended to include scFv fragments including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This will be apparent to those skilled in the art.

본 발명에서 용어, "상보성결정영역(complementarity determining region, CDR)"은 면역글로불린의 중쇄 및 경쇄의 고가변 영역 (hypervariable region)의 아미노산 서열을 의미한다. 중쇄 및 경쇄는 각각 3개의 CDR을 포함할 수 있다 (CDRH1, CDRH2,CDRH3 및 CDRL1, CDRL2, CDRL3). 상기 CDR은 항체가 항원 또는 항원결정부위에 결합하는 데 있어서 주요한 접촉 잔기를 제공할 수 있다. In the present invention, the term "complementarity determining region (CDR)" refers to an amino acid sequence of a hypervariable region of a heavy chain and a light chain of an immunoglobulin. The heavy chain and the light chain may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for binding of an antibody to an antigen or an epitope.

본 발명에서, 용어, "특이적으로 결합" 또는 "특이적으로 인식"은 당업자에게 통상적으로 공지되어 있는 의미와 동일한 것으로서, 항원 및 항체가 특이적으로 상호작용하여 면역학적 반응을 하는 것을 의미한다.In the present invention, the terms “specifically bind” or “specifically recognize” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.

본 발명에서 용어, "항원결합단편"은 면역글로불린 전체 구조에 대한 그의 단편으로, 항원이 결합할 수 있는 부분을 포함하는 폴리펩타이드의 일부를 의미한다. 예를 들어, scFv, (scFv) 2 , scFv-Fc, Fab, Fab' 또는 F(ab') 2 일 수 있으나, 이에 한정되지 않는다. 상기 항원결합단편 중 Fab는 경쇄 및 중쇄의 가변 영역과 경쇄의 불변 영역 및 중쇄의 첫 번째 불변 영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab') 2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변 영역 및 경쇄 가변 부위만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 당업계에 널리 공지되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변 부위와 경쇄 가변 부위가 연결되어 있고 단쇄 Fv(single-chain Fv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변 영역과 단쇄의 가변 영역이 공유 결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 상기 링커는 1 내지 100개 또는 2 내지 50개의 임의의 아미노산으로 이루어진 펩타이드 링커일 수 있으며, 당업계에 적절한 서열이 알려져 있다. 상기 항원결합단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab') 2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수 있다.In the present invention, the term "antigen-binding fragment" refers to a fragment of the entire structure of an immunoglobulin, and means a part of a polypeptide including a portion capable of binding an antigen. For example, it may be scFv, (scFv) 2 , scFv-Fc, Fab, Fab' or F(ab') 2 , but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having variable regions of light and heavy chains, a constant region of a light chain, and a first constant region (C H1 ) of a heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain C H1 domain. F(ab') 2 antibody is produced when the cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant technology for producing Fv fragments is widely known in the art. A two-chain Fv has a heavy chain variable region and a light chain variable region linked by a non-covalent bond, and a single-chain Fv generally has a heavy chain variable region and a single chain variable region linked by a covalent bond or directly at the C-terminus via a peptide linker to form a dimer structure like a two-chain Fv. The linker may be a peptide linker composed of any amino acid of 1 to 100 or 2 to 50, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a protein hydrolase (for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and a F(ab') 2 fragment can be obtained by digestion with pepsin), or can be produced by a genetic recombination technique.

본 발명에서 용어 "힌지 영역(hunge region)"은 항체의 중쇄에 포함되어 있는 영역으로서, CH1 및 CH2 영역 사이에 존재하며, 항체 내 항원 결합 부위의 유연성(flexibility)를 제공하는 기능을 하는 영역을 의미한다. 예컨대, 상기 힌지는 인간 항체로부터 유래한 것일 수 있으며, 구체적으로, IgA, IgE, 또는 IgG, 예컨대, IgG1, IgG2, IgG 3, 또는 IgG4로부터 유래한 것일 수 있다.The term "hinge region" in the present invention refers to a region included in the heavy chain of an antibody, which exists between the C H1 and C H2 regions and has the function of providing flexibility to the antigen binding site within the antibody. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, for example, IgG1, IgG2, IgG 3, or IgG4.

본 발명에서 용어, "아미노산 변형/변이"는 폴리펩타이드 서열의 아미노산의 치환, 삽입 및/또는 결실, 바람직하게는 치환을 의미한다. 본 발명에서 사용된, 용어 "아미노산 치환" 또는 "치환"은 항체의 폴리펩타이드 서열의 특정 위치에서의 아미노산이 다른 아미노산으로 대체되는 것을 의미한다.As used herein, the term "amino acid modification/mutation" means substitution, insertion and/or deletion, preferably substitution, of an amino acid in a polypeptide sequence. As used herein, the term "amino acid substitution" or "substitution" means that an amino acid at a specific position in a polypeptide sequence of an antibody is replaced with another amino acid.

본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 당해 기술분야에 공지된 임의의 방법으로 제조될 수 있다. 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편의 폴리펩타이드 서열을 코딩한 후, 원하는 경우, 숙주세포 내로 클로닝되고, 발현 및 검정되는 핵산 형성에 이용된다. 이를 위한 다양한 방법이 문헌 (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons)에 기재되어 있다.The antibody of the present invention or a fragment thereof having immunological activity can be prepared by any method known in the art. After encoding the polypeptide sequence of the antibody of the present invention or a fragment thereof having immunological activity, it is used to form a nucleic acid that is cloned into a host cell and expressed and assayed, if desired. Various methods for this are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).

본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산은 단백질 발현을 위해 발현벡터에 삽입될 수 있다. 발현벡터는, 통상 조절 또는 제어(regulatory) 서열, 선별마커, 임의의 융합 파트너, 및/또는 추가적 요소와 작동가능하게 연결된, 즉, 기능적 관계에 놓인 단백질을 포함한다. 적절한 상태에서, 핵산으로 형질전환된 숙주세포, 바람직하게는, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산 함유 발현벡터를 배양하여 단백질 발현을 유도하는 방법에 의해 본 발명에 따른 항체 또는 이의 면역학적 활성을 가진 단편이 생산될 수 있다. 포유류 세포, 박테리아, 곤충 세포, 및 효모를 포함하는 다양한 적절한 숙주세포가 사용될 수 있으나, 이에 제한하는 것은 아니다. 외인성 핵산을 숙주세포에 도입하는 방법은 당해 기술분야에 공지되어 있으며, 사용되는 숙주세포에 따라 달라질 것이다. 바람직하게는, 생산비가 저렴하여 산업적 이용가치가 높은 대장균을 숙주세포로 하여 본 발명에 따른 항체 또는 이의 면역학적 활성을 가진 단편을 생산한다.The nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity can be inserted into an expression vector for protein expression. The expression vector typically includes a protein operably linked, i.e., in a functional relationship, with a regulatory sequence, a selectable marker, an optional fusion partner, and/or additional elements. Under appropriate conditions, the antibody according to the present invention or a fragment thereof having immunological activity can be produced by a method of culturing a host cell transformed with the nucleic acid, preferably an expression vector containing a nucleic acid encoding the antibody of the present invention or a fragment thereof having immunological activity, to induce protein expression. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, the antibody according to the present invention or a fragment thereof having immunological activity is produced using E. coli, which has low production costs and high industrial utility, as a host cell.

따라서, 본 발명의 범위에는 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산이 도입된 숙주세포를 단백질 발현에 적합한 조건 하에서 배양하는 단계; 및 숙주세포로부터 발현된 항체 또는 이의 면역학적 활성을 가진 단편을 정제 또는 분리하는 단계를 포함하는 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편의 제조방법이 포함된다.Therefore, the scope of the present invention includes a method for producing an antibody or a fragment having immunological activity specific for EphA10, comprising the steps of: culturing a host cell into which a nucleic acid encoding the antibody or a fragment having immunological activity thereof of the present invention has been introduced under conditions suitable for protein expression; and purifying or isolating the antibody or fragment having immunological activity thereof expressed from the host cell.

항체는 당해 기술분야에서 공지된 다양한 방법으로 분리 또는 정제될 수 있다. 표준 정제방법은 크로마토그래피 기술, 전기영동, 면역, 침강, 투석, 여과, 농축, 및 크로마토포커싱 (chromatofocusing) 기술을 포함한다. 당해 기술분야에 공지된 바와 같이, 예를 들어 박테리아 단백질 A, G, 및 L과 같은 다양한 천연 단백질이 항체와 결합하며, 상기 단백질은 정제에 이용될 수 있다. 종종, 특정 융합 파트너에 의한 정제가 가능할 수 있다. Antibodies can be isolated or purified by a variety of methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunohistochemistry, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, a variety of natural proteins bind antibodies, such as, for example, bacterial proteins A, G, and L, and such proteins can be used for purification. Often, purification by specific fusion partners can be achieved.

일 측면에서, 본 발명은 본 발명의 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편, 및 약물을 포함하는, 항체-약물 접합체(Antibody-Drug Conjugate, ADC)에 관한 것이다.In one aspect, the present invention relates to an antibody-drug conjugate (ADC), comprising an antibody specific for EphA10 of the present invention or a fragment thereof having immunological activity, and a drug.

일 구현예에서, 약물은 약물은 면역원성 세포사멸 유도제, 전세포사멸성(pro-apoptotic) 펩타이드, 이크로튜불린(microtubulin) 구조 형성 억제제, 유사분열(meiosis) 억제제, 토포아이소머라아제(topoisomerase) 억제제, DNA 인터컬레이터(DNA intercalators), 독소(toxin), 방사선 핵종 또는 항암제일 수 있다.In one embodiment, the drug may be an immunogenic apoptosis inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a radionuclide, or an anticancer agent.

일 구현예에서, 상기 전세포사멸성 펩타이드는 KLA, 알파-디펜신-1(alpha-defensin-1), BMAP-28, Brevenin-2R, 부포린 IIb(Buforin IIb), 세크로핀 A-마가이닌 2(cecropin A-Magainin 2, CA-MA-2), 세크로핀 A(Cecropin A), 세크로핀 B(Cecropin B), 크리소피신-1(chrysophsin-1), D-K6L9, 고메신(Gomesin), 락토페리신 B(Lactoferricin B), LLL27, LTX-315, 마가이닌 2(Magainin 2), 마가이닌 II-봄패신 결합체(Magainin II-bombesin conjugate, MG2B), 파르닥신(Pardaxin) 및 이들의 조합으로 이루어진 군에서 선택될 수 있다.In one embodiment, the pro-apoptotic peptide can be selected from the group consisting of KLA, alpha-defensin-1, BMAP-28, Brevenin-2R, Buforin IIb, cecropin A-Magainin 2 (CA-MA-2), Cecropin A, Cecropin B, chrysophsin-1, D-K6L9, Gomesin, Lactoferricin B, LLL27, LTX-315, Magainin 2, Magainin II-bombesin conjugate (MG2B), Pardaxin, and combinations thereof.

일 구현예에서, 상기 면역원성 세포사멸 유도제는 안트라사이클린계열 항암제, 탁산 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside), 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제, LV-tSMAC, Measles 바이러스, 블레오마이신(bleomycin), 미토잔트론(mitoxantrone), 옥살리플라틴(oxaliplatin) 및 이들의 조합으로 이루어진 군에서 선택될 수 있다.In one embodiment, the immunogenic apoptosis inducer can be selected from the group consisting of anthracycline anticancer agent, taxane anticancer agent, anti-EGFR antibody, BK channel agonist, bortezomib, cardiac glycoside, cyclophosphamide anticancer agent, GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, oxaliplatin, and combinations thereof.

일 구현예에서, 상기 항암제는 SN-38(7-에틸-10-히드록시-캠토테신, 7-Ethyl-10-hydroxy-camptothecin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin), 발루비신(valrubicin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메클로에타민(mechloethamine), 클로람부실(chlorambucil), 페닐알라닌(phenylalanine), 무스타드(mustard), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 카르무스틴(carmustine: BCNU), 로무스틴(lomustine: CCNU), 스트렙토조토신(streptozotocin), 부설판(busulfan), 티오테파(thiotepa), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 닥티노마이신(dactinomycin: actinomycin D), 플리카마이신(plicamycin), 마이토마이신 C(mitomycin C), 빈크리스틴(vincristine), 빈블라스틴(vinblastine), 테니포사이드(teniposide), 토포테칸(topotecan), 이리도테칸(iridotecan), 우라무스틴(uramustine), 멜파란(melphalan), 벤다무스틴(bendamustine), 다카바진(dacarbazine), 테모졸로마이드(temozolomide), 알트레타민(altretamine), 듀오카르마이신(duocarmycin), 네다플라틴(nedaplatin), 옥사리플라틴(oxaliplatin), 사트라플라틴(satraplatin), 트리플라틴 테트라나이트레이트(triplatin tetranitrate), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurine), 카페시타빈(capecitabine), 클라드리빈(cladribine), 클로파라빈(clofarabine), 시스타르빈(cystarbine), 플록스유리딘(floxuridine), 플루다라빈(fludarabine), 겜시타빈(gemcitabine), 하이드록시우레아(hydroxyurea), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 펜토스타틴(pentostatin), 티오구아닌(thioguanine), 에토포사이드(etoposide), 미토산트론(mitoxantrone), 이자베필론(izabepilone), 빈데신(vindesine), 비노렐빈(vinorelbine), 에스트라머스틴(estramustine), 메이탄신(maytansine), DM1(mertansine, 메르탄신), DM4, 돌라스타틴(dolastatin), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E, MMAE), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군에서 선택될 수 있다.In one embodiment, the anticancer agent is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, It may be selected from the group consisting of maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

일 구현예에서, 상기 항암제는 면역항암제일 수 있으며, 면역항암제는 면역관문 억제제(Immune checkpoint inhibitors), 면역억제인자 제어 약물, 암 백신, 면역아주번트(immunoadjuvant), 암 치료용 면역세포, 면역세포 활성 보조인자, 암 치료용 항체 또는 암 치료용 면역세포의 활성 유지에 필요한 사이토카인일 수 있다. In one embodiment, the anticancer agent may be an immunotherapy agent, and the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant control drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.

일 구현예에서, 상기 면역억제인자 제어 약물은 조절 T 세포(regulatory T cells, Treg)의 수준을 감소시키는 약물일 수 있으며, 조절 T 세포를 감소시킴으로써 T 작동 세포(T effector cells)의 면역 반응을 활성화시킬 수 있다.In one embodiment, the immunosuppressant controlling drug may be a drug that reduces the level of regulatory T cells (Treg), and thereby activates the immune response of T effector cells.

일 구현예에서, 상기 면역관문 억제제는 CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA 또는 A2aR의 억제제일 수 있으며, 항-PD-1 항체, 항-PD-L1 항체, 항-CTLA-4 항체, 또는 이의 변이체일 수 있다.In one embodiment, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR, and can be an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or a variant thereof.

일 구현예에서, 약물은 SN-38(7-에틸-10-히드록시-캠토테신, 7-Ethyl-10-hydroxy-camptothecin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin), 발루비신(valrubicin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메클로에타민(mechloethamine), 클로람부실(chlorambucil), 페닐알라닌(phenylalanine), 무스타드(mustard), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 카르무스틴(carmustine: BCNU), 로무스틴(lomustine: CCNU), 스트렙토조토신(streptozotocin), 부설판(busulfan), 티오테파(thiotepa), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 닥티노마이신(dactinomycin: actinomycin D), 플리카마이신(plicamycin), 마이토마이신 C(mitomycin C), 빈크리스틴(vincristine), 빈블라스틴(vinblastine), 테니포사이드(teniposide), 토포테칸(topotecan), 이리도테칸(iridotecan), 우라무스틴(uramustine), 멜파란(melphalan), 벤다무스틴(bendamustine), 다카바진(dacarbazine), 테모졸로마이드(temozolomide), 알트레타민(altretamine), 듀오카르마이신(duocarmycin), 네다플라틴(nedaplatin), 옥사리플라틴(oxaliplatin), 사트라플라틴(satraplatin), 트리플라틴 테트라나이트레이트(triplatin tetranitrate), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurine), 카페시타빈(capecitabine), 클라드리빈(cladribine), 클로파라빈(clofarabine), 시스타르빈(cystarbine), 플록스유리딘(floxuridine), 플루다라빈(fludarabine), 겜시타빈(gemcitabine), 하이드록시우레아(hydroxyurea), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 펜토스타틴(pentostatin), 티오구아닌(thioguanine), 에토포사이드(etoposide), 미토산트론(mitoxantrone), 이자베필론(izabepilone), 빈데신(vindesine), 비노렐빈(vinorelbine), 에스트라머스틴(estramustine), 메이탄신(maytansine), DM1(mertansine, 메르탄신), DM4, 돌라스타틴(dolastatin), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E, MMAE), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군으로부터 선택될 수 있다.In one embodiment, the drug is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, It may be selected from the group consisting of maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof.

일 구현예에서, 상기 항체-약물 접합체는 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편과 약물이 링커를 매개로 연결/결합될 수 있으며, 링커는 펩타이드나 리간드, 항체, 항체 단편 등의 단백질의 아민기(amine group), 카르복시기(carboxyl group) 또는 설프히드릴기(sulfhydryl group)나 압타머 등의 핵산의 인산기(phosphate group, 히드록시기(hydroxyl group)를 통해 결합할 수 있는 작용기를 가진 임의의 링커를 사용할 수 있다. 이러한 링커의 작용기는 아이소티오시아네이트(isothiocyanate), 아이소시아네이트(isocyanates), 아실 아자이드(acyl azide), NHS 에스터(NHS ester), 설포닐 클로라이드(sulfonyl chloride), 알데하이드(aldehyde), 글리옥살(glyoxal), 에폭사이드(epoxide), 옥시레인(oxirane), 칼보네이트(carbonate), 아릴 할라이드(arylhalide), 이미도에스터(imidoester), 카보이미드(carbodiimide), 안하이드라이드(anhydride), 플루오로페닐 에스터(fluorophenyl ester), 히드록시메틸포스핀(hydroxymethyl phosphine), 말레이미드(maleimide), 할로아세틸(haloacetyl), 피리딜디설파이드(pyridyldisulfide), 티오술포네이트(thiosulfonate), 또는 비닐술폰(vinylsulfone) 등일 수 있다. 링커는 프로테아제에 의해서 절단 가능하거나, 산이나 염기 조건에서 절단 가능하거나, 고온이나 광조사에 의해서 절단 가능하거나 또는 환원 또는 산화 조건에서 절단 가능한 링커일 수 있고, 또는 이러한 조건들에서 절단 가능하지 않은 링커일 수도 있다. 절단 가능한 링커로서는 예컨대 산성 조건에서 절단되는 히드라존(hydrazone) 링커, 프로테아제에 의해 절단되는 펩타이드 링커, 환원 조건에서 절단되는 디설파이드(disulfide) 작용기를 갖는 링커 등을 들 수 있고, 절단 가능하지 않은 링커로서는 MCC(Maleimidomethyl cyclohexane-1-carboxylate) 링커, MC(maleimidocaproyl) 링커, 또는 그 유도체로서 석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sMCC) 링커나 설포석신이미딜-4-(N-말레이미도메틸)사이클로헥산-1-카르복실레이트(sulfo-sMCC)를 들 수 있다. In one embodiment, the antibody-drug conjugate may be connected/bound to the antibody of the present invention or a fragment thereof having immunological activity and the drug via a linker, and the linker may be any linker having a functional group capable of binding to an amine group, a carboxyl group, or a sulfhydryl group of a protein such as a peptide, a ligand, an antibody, or an antibody fragment, or a phosphate group, a hydroxyl group of a nucleic acid such as an aptamer. The functional group of such a linker may be isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, The linker may be an imidoester, a carbodiimide, an anhydride, a fluorophenyl ester, a hydroxymethyl phosphine, a maleimide, a haloacetyl, a pyridyldisulfide, a thiosulfonate, or a vinylsulfone. The linker may be a linker that is cleavable by a protease, a linker that is cleavable under acid or base conditions, a linker that is cleavable by high temperature or light irradiation, a linker that is cleavable under reducing or oxidizing conditions, or a linker that is not cleavable under these conditions. Cleavable linkers include, for example, a hydrazone linker that is cleavable under acidic conditions, a peptide linker that is cleavable by a protease, a linker having a disulfide functional group that is cleavable under reducing conditions, and a non-cleavable linker includes, for example, Examples of the linker include MCC (Maleimidomethyl cyclohexane-1-carboxylate), MC (maleimidocaproyl) linker, or derivatives thereof such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) linker or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).

또한 링커는 자가 희생 링커(self-immolative linker) 또는 절단 후 흔적을 남기지 않는 링커(traceless linker)일 수 있다. 자가 희생 링커는 예컨대 발명의 명칭이 "Hydrophilic self-immolative linkers and conjugates thereof "인 미국 특허 제9,089,614호에 개시된 링커, 명칭이 "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF"인 국제공개 제WO2015038426호에 개시된 링커를 들 수 있으며, 절단 후 흔적을 남기지 않는 링커로서는 페닐하이드라지드 링커, 아릴-트리아젠 링커, 문헌[Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. 102: 2607-2024 (2002)]에 개시된 링커 등일 수 있다.Additionally, the linker may be a self-immolative linker or a traceless linker. Self-immolative linkers include, for example, linkers disclosed in U.S. Pat. No. 9,089,614 entitled "Hydrophilic self-immolative linkers and conjugates thereof," linkers disclosed in International Publication No. WO2015038426 entitled "SELF-IMMOLATIVE LINKERS CONTAINING MANDELIC ACID DERIVATIVES, DRUG-LIGAND CONJUGATES FOR TARGETED THERAPIES AND USES THEREOF," and traceless linkers include, for example, phenylhydrazide linkers, aryl-triazene linkers, and linkers disclosed in the literature [Blaney, et al., "Traceless solid-phase organic synthesis," Chem Rev. It may be a linker, etc. disclosed in [102: 2607-2024 (2002)].

본 발명의 항체-약물 접합체의 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편과 약물은 생체 적합성 고분자를 매개로 또는 캐리어로 하여 결합될 수도 있다. 생체적합성 고분자는 생체조직 또는 혈액과 접촉하여 조직을 괴사시키거나 혈액을 응고시키지 않는 조직적합성(tissue compatibility) 및 항응혈성(blood compatibility)을 가지고 있는 고분자를 의미한다. 상기 생체적합성 고분자로서의 합성 중합체는 폴리에스테르, 폴리하이드 록시알카노에이트(PHAs), 폴리(α-하이드록시액시드), 폴리(β-하이드록시액시드), 폴리(3-하이드로식부티레이트-co-발러레이트; PHBV), 폴리(3-하이드록시프로프리오네이트; PHP), 폴리(3-하이드록시헥사노에이트; PHH), 폴리(4-하이드록시액시드), 폴리(4-하이드록시부티레이트), 폴리(4-하이드록시발러레이트), 폴리(4-하이드록시헥사노에이트), 폴리(에스테르아마이드), 폴리카프로락톤, 폴리락타이드, 폴리글리코라이드, 폴리(락타이드-co-글리코라이드; PLGA), 폴리디옥사논, 폴리오르토에스테르, 폴리언하이드라이드, 폴리(글리콜산-co-트리메틸렌카보네이트), 폴리포스포에스테르, 폴리포스포에스테르 우레탄, 폴리(아미노산), 폴리사이아노아크릴레이트, 폴리(트리메틸렌 카보네이트), 폴리(이미노카보네이트), 폴리(타이로신 카보네이트), 폴리카보네이트, 폴리(타이로신 아릴레이트), 폴리알킬렌 옥살레이트, 폴리포스파젠스, PHA-PEG, 에틸렌 비닐 알코올 코폴리머(EVOH), 폴리우레탄, 실리콘, 폴리에스테르, 폴리올레핀, 폴리이소부틸렌과 에틸렌-알파올레핀 공중합체, 스틸렌-이소브틸렌-스틸렌 트리블록 공중합체, 아크릴 중합체 및 공중합체, 비닐 할라이드 중합체 및 공중합체, 폴리비닐 클로라이드, 폴리비닐 에테르, 폴리비닐 메틸에테르, 폴리비닐리덴 할라이드, 폴리비닐리덴 플루오라이드, 폴리비닐리덴 클로라이드, 폴리플루오로알켄, 폴리퍼플루오로알켄, 폴리아크릴로니트릴, 폴리비닐 케톤, 폴리비닐 아로마틱스, 폴리스틸렌, 폴리비닐 에스테르, 폴리비닐 아세테이트, 에틸렌-메틸 메타크릴레이트 공중합체, 아크릴로니트릴-스틸렌 공중합체, ABS 수지와 에틸렌-비닐 아세테이트 공중합체, 폴리아마이드, 알키드 수지, 폴리옥시메틸렌, 폴리이미드, 폴리에테르, 폴리아크릴레이트, 폴리메타크릴레이트, 폴리아크릴산-co-말레산 또는 폴리아미노아민이며, 천연 중합체는 키토산, 덱스트란, 셀룰로오스, 헤파린, 히알루론산, 알기네이트, 이눌린, 녹말 또는 글리코겐이다.The antibody of the present invention or a fragment thereof having immunological activity and the drug of the antibody-drug conjugate of the present invention may be combined via a biocompatible polymer or a carrier. A biocompatible polymer means a polymer having tissue compatibility and blood compatibility that does not cause necrosis of tissue or coagulation of blood upon contact with living tissue or blood. Synthetic polymers as the biocompatible polymers include polyesters, polyhydroxyalkanoates (PHAs), poly(α-hydroxyacids), poly(β-hydroxyacids), poly(3-hydroxybutyrate-co-valerate; PHBV), poly(3-hydroxyproprionate; PHP), poly(3-hydroxyhexanoate; PHH), poly(4-hydroxyacids), poly(4-hydroxybutyrate), poly(4-hydroxyvalerate), poly(4-hydroxyhexanoate), poly(esteramide), polycaprolactone, polylactide, polyglycolide, poly(lactide-co-glycolide; PLGA), polydioxanone, polyorthoester, polyanhydride, poly(glycolic acid-co-trimethylene carbonate), polyphosphoester, polyphosphoester urethane, poly(amino acid), Polycyanoacrylates, poly(trimethylene carbonate), poly(iminocarbonate), poly(tyrosine carbonate), polycarbonates, poly(tyrosine arylate), polyalkylene oxalates, polyphosphazenes, PHA-PEG, ethylene vinyl alcohol copolymers (EVOH), polyurethanes, silicones, polyesters, polyolefins, polyisobutylene and ethylene-alphaolefin copolymers, styrene-isobutylene-styrene triblock copolymers, acrylic polymers and copolymers, vinyl halide polymers and copolymers, polyvinyl chloride, polyvinyl ethers, polyvinyl methyl ether, polyvinylidene halides, polyvinylidene fluoride, polyvinylidene chloride, polyfluoroalkenes, polyperfluoroalkenes, polyacrylonitrile, polyvinyl ketones, polyvinyl aromatics, polystyrene, polyvinyl esters, polyvinyl acetate, Ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin and ethylene-vinyl acetate copolymer, polyamide, alkyd resin, polyoxymethylene, polyimide, polyether, polyacrylate, polymethacrylate, polyacrylic acid-co-maleic acid or polyaminoamine, and natural polymers are chitosan, dextran, cellulose, heparin, hyaluronic acid, alginate, inulin, starch or glycogen.

일 구현예에서, 상기 항체-약물 접합체는 ADC 링커를 추가로 포함할 수 있으며, ADC 링커는 6-말레이미도카프로일(MC), 말레이미도프로파노일(MP), 발린-시트룰린(val-cit), 알라닌-페닐알라닌(ala-phe), p-아미노벤질옥시카르보닐(PAB), N-숙신이미딜 4-(2-피리딜티오) 펜타노에이트(SPP), N-숙신이미딜 4-(N-말레이미도메틸) 시클로헥산-1 카르복실레이트(SMCC), 발린-시트룰린-p-아미노벤질옥시카르보닐(val-cit-PAB) 또는 N-숙신이미딜 (4-요오도-아세틸) 아미노벤조에이트(SIAB)일 수 있다.In one embodiment, the antibody-drug conjugate can further comprise an ADC linker, which can be 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

일 구현예에서, 상기 항체-약물 접합체는 ADC 링커를 통해 본 발명의 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편이 약물과 복합체를 형성할 수 있다.In one embodiment, the antibody-drug conjugate can form a complex with an antibody specific for EphA10 of the present invention or a fragment thereof having immunological activity, through an ADC linker.

일 구현예에서, 본 발명의 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편에 RNA, DNA, 항체, 이펙터, 약물, 전구약물, 독소, 펩타이드 또는 전달 분자가 추가로 접합(conjugate)될 수 있다 (Shoari et al., Pharmaceutics 13:1391, pp. 1-32 (2021) 참조).In one embodiment, an antibody specific for EphA10 of the present invention or an immunologically active fragment thereof may be additionally conjugated to an RNA, DNA, antibody, effector, drug, prodrug, toxin, peptide or delivery molecule (see Shoari et al., Pharmaceutics 13:1391, pp. 1-32 (2021)).

일 측면에서, 본 발명은 본 발명의 EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편, 및 EphA10 이외의 표적 항원에 결합하는 부분을 포함하는 이중특이적 또는 다중특이적 항체에 관한 것이다.In one aspect, the present invention relates to a bispecific or multispecific antibody comprising an antibody specific for EphA10 of the present invention or a fragment having immunological activity thereof, and a portion that binds to a target antigen other than EphA10.

일 구현예에서, 표적 항원에 결합하는 부분이 항체 또는 이의 면역학적 활성을 가진 단편을 포함할 수 있다.In one embodiment, the moiety that binds to the target antigen may comprise an antibody or a fragment having immunological activity thereof.

일 구현예에서, 표적 항원은 17-1A 항원, GD3 갱글리오시드 R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 코어 단백질, 이상 글리코실화 MUC1, ED-B 도메인을 함유하는 피브로넥틴 이형, HER2/neu, 암종배아성 항원(CEA), 가스트린-방출 펩티드(GRP) 수용체 항원, 뮤신(mucine) 항원, 표피 성장 인자 수용체(EGF-R), HER3, HER4, MAGE 항원, SART 항원, MUC1 항원, c-erb-2 항원, TAG 72, 탄산 무수화효소 IX(carbonic anhydrase IX), 알파-태아단백질(alpha-fetoprotein), A3, A33 항체에 특이적인 항원, Ba 733, BrE3-항원, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, 결장-특이적 항원-p(CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, 폴산염 수용체(folate receptor), HLA-DR, 인간 융모성 성선자극호르몬(HCG) 및 그 소단위체들, 저산소증 유도 인자(HIF-I), Ia, IL-2, IL-6, IL-8, 인슐린 성장 인자-1(IGF-1), KC4-항원, KS-1-항원, KSl-4, Le-Y, 대식세포 억제 인자(MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM-4 항체에 특이적인 항원, 태반 성장 인자, p53, 전립선 산성 포스파타제(prostatic acid phosphatase), PSA, RS5, SlOO, TAC, 테나신(tenascin), TRAIL 수용체들, Tn 항원, 톰슨-프리덴라이히(Thomson-Friedenreich) 항원들, 종양 괴사 항원들, VEGF, ED-B 피브로넥틴(fibronectin), 혈관형성 표지(angiogenesis marker), 종양유전자 표지(oncogene marker) 또는 종양유전자 생성물로 이루어지는 군으로부터 선택되는 어느 하나 이상일 수 있다.In one embodiment, the target antigen is an antigen specific for 17-1A antigen, GD3 ganglioside R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrantly glycosylated MUC1, fibronectin isoform containing ED-B domain, HER2/neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, A33 antibody, Ba 733, BrE3-antigen, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KSl-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, It may be at least one selected from the group consisting of antigens specific for NCA66, NCA95, NCA90, PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SlOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, angiogenesis marker, oncogene marker or oncogene product.

일 구현예에서, 상기 아폽토시스 관련 유전자는 ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ 또는 YWHAZ일 수 있으며; 상기 전사인자 유전자는 AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217 또는 ZNF268일 수 있고; 상기 전이 관련 유전자는 AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3 또는 WT1일 수 있으며; 상기 혈관신생 관련 유전자는 BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB 또는 SPARC일 수 있고; 상기 티로신-카이네이즈 유전자는 ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC 또는 YES1일 수 있다.In one embodiment, the apoptosis-related genes are ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, It can be PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ or YWHAZ; The transcription factor genes are AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, 4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, , MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP 63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217 or ZNF268; The above metastasis-related gene may be AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3 or WT1; the above angiogenesis-related gene may be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB or SPARC; The above tyrosine-kinase gene can be ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC or YES1.

일 구현예에서, 상기 종양유전자는 SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ, GNAS, GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1, PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIR, PIWIL1, PLAC8, PLK1, PPM1D, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, PVT1, RAB11A, RAB18, RAB22A, RAB23, RAB8A, RALGDS, RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF1, RUNX1T1, S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, XIAP, YAP1, YEATS4, YY1AP1, ZEB1-AS1, ZFAND4, ZFAS1, ZMYM2, ZNF703 및 ZNHIT6로 이루어지는 군으로부터 선택되는 어느 하나 이상일 수 있다.In one embodiment, the oncogene is SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1 , ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D , CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, , EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ, GNAS, GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1, PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIR, PIWIL1, PLAC8, PLK1, PPM1D, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, PVT1, RAB11A, RAB18, RAB22A, RAB23, RAB8A, RALGDS, RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF1, RUNX1T1, S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, With ZFAS1, ZMYM2, ZNF703 and ZNHIT6 It can be one or more selected from the group consisting of:

일 구현예에서, 표적 항원은 세포 표면 항원 또는 자가항원일 수 있다.In one embodiment, the target antigen can be a cell surface antigen or an autoantigen.

일 구현예에서, 상기 세포 표면 항원은 CEA, ED-B 피브로넥틴, CD20, CD22, CDl9, EGFR, IGFlR, VEFGRl/Flt-1, VEGFR2/KDR, VEGRF3/Flt-4, HER2/neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, 아데노바이러스 섬유 놉, PfMSP-1, HN/NDV, EpCAM/17-lA, hTR, IL-2R/Tac, CAl9-9, MUCl, HLA 클래스 II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, Ml3 외피 단백질 및 GPIIb/IIIa로 이루어지는 군으로부터 선택되는 어느 하나 이상일 수 있다.In one embodiment, the cell surface antigen can be at least one selected from the group consisting of CEA, ED-B fibronectin, CD20, CD22, CDl9, EGFR, IGFlR, VEFGRl/Flt-1, VEGFR2/KDR, VEGRF3/Flt-4, HER2/neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fiber knob, PfMSP-1, HN/NDV, EpCAM/17-lA, hTR, IL-2R/Tac, CA19-9, MUCl, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, Ml3 coat protein, and GPIIb/IIIa.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 또는 본 발명의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자, 이를 포함하는 벡터, 및 이로 형질전환된 숙주 세포에 관한 것이다.In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment having immunological activity thereof, or a bispecific or multispecific antibody of the present invention, a vector comprising the same, and a host cell transformed therewith.

본 발명의 핵산 분자는 단리된 것이거나 재조합된 것일 수 있으며, 단일쇄 및 이중쇄 형태의 DNA 및 RNA뿐만 아니라 대응하는 상보성 서열이 포함된다. 단리된 핵산은 천연 생성 원천에서 단리된 핵산의 경우, 핵산이 단리된 개체의 게놈에 존재하는 주변 유전 서열로부터 분리된 핵산이다. 주형으로부터 효소적으로 또는 화학적으로 합성된 핵산, 예컨대 PCR 산물, cDNA 분자, 또는 올리고뉴클레오타이드의 경우, 이러한 절차로부터 생성된 핵산이 단리된 핵산분자로 이해될 수 있다. 단리된 핵산분자는 별도 단편의 형태 또는 더 큰 핵산 구축물의 성분으로서의 핵산 분자를 나타낸다. 핵산은 다른 핵산 서열과 기능적 관계로 배치될 때 작동가능하게 연결된다. 예를 들면, 전서열 또는 분비 리더(leader)의 DNA는 폴리펩타이드가 분비되기 전의 형태인 전단백질(preprotein)로서 발현되는 경우 폴리펩타이드의 DNA에 작동가능하게 연결되고, 프로모터 또는 인핸서는 폴리펩타이드 서열의 전사에 영향을 주는 경우 코딩 서열에 작동가능하게 연결되며, 또는 리보솜 결합 부위는 번역을 촉진하도록 배치될 때 코딩 서열에 작동가능하게 연결된다. 일반적으로 작동가능하게 연결된은 연결될 DNA 서열들이 인접하여 위치함을 의미하며, 분비 리더의 경우 인접하여 동일한 리딩 프레임 내에 존재하는 것을 의미한다. 그러나 인핸서는 인접하여 위치할 필요는 없다. 연결은 편리한 제한 효소 부위에서 라이게이션에 의해 달성된다. 이러한 부위가 존재하지 않는 경우, 합성 올리고뉴클레오타이드 어댑터 또는 링커를 통상적인 방법에 따라 사용한다.The nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that is separated from the surrounding genetic sequence present in the genome of the organism from which the nucleic acid is isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a procedure may be understood to be an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a pre-sequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein, that is, a form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence when it is positioned so as to facilitate translation. Operably linked generally means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in a conventional manner.

본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 또는 본 발명의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자는 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 항체를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 항체의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 핵산 분자는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. 이러한 핵산 분자의 서열은 단쇄 또는 이중쇄일 수 있으며, DNA 분자 또는 RNA(mRNA)분자일 수 있다.The isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention may have various modifications in the coding region within a range that does not change the amino acid sequence of the antibody expressed from the coding region due to the degeneracy of the codon or in consideration of the codon preferred in the organism to which the antibody is to be expressed, and various modifications or alterations may be made in a portion excluding the coding region within a range that does not affect the expression of the gene, and it will be well understood by those skilled in the art that such modified genes are also included in the scope of the present invention. That is, the nucleic acid molecule of the present invention may have one or more nucleic acid bases mutated by substitution, deletion, insertion, or a combination thereof, as long as it encodes a protein having an activity equivalent thereto, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

본 발명에 따른 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 또는 본 발명의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자는 단백질 발현을 위해 발현벡터에 삽입될 수 있다. 발현벡터는, 통상 조절 또는 제어 (regulatory) 서열, 선별마커, 임의의 융합 파트너, 및/또는 추가적 요소와 작동가능하게 연결된, 즉, 기능적 관계에 놓인 단백질을 포함한다. 적절한 상태에서, 핵산으로 형질전환된 숙주세포, 바람직하게는, 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 또는 본 발명의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자 함유 발현벡터를 배양하여 단백질 발현을 유도하는 방법에 의해 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 또는 본 발명의 이중특이적 또는 다중특이적 항체가 생산될 수 있다. 포유류 세포, 박테리아, 곤충 세포, 및 효모를 포함하는 다양한 적절한 숙주세포가 사용될 수 있으나, 이에 제한하는 것은 아니다. 외인성 핵산을 숙주세포에 도입하는 방법은 당해 기술분야에 공지되어 있으며, 사용되는 숙주세포에 따라 달라질 것이다. 바람직하게는, 생산비가 저렴하여 산업적 이용가치가 높은 대장균을 숙주세포로 생산할 수 있다.The isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention can be inserted into an expression vector for protein expression. The expression vector typically comprises the protein operably linked, i.e., in a functional relationship, with a regulatory or control sequence, a selectable marker, an optional fusion partner, and/or additional elements. Under appropriate conditions, the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention can be produced by a method of culturing a host cell transformed with the nucleic acid, preferably an expression vector containing the isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity, or the bispecific or multispecific antibody of the present invention, to induce protein expression. Various suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, E. coli, which has a low production cost and high industrial utility value, can be used as the host cell.

본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 상기 시그널 서열에는 숙주가 에쉐리키아속(Escherichia sp.)균인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속(Bacillus sp.)균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모(yeast)인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널 서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다. 또한 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고, 복제 가능한 발현벡터인 경우 복제 기원을 포함한다.The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. A suitable vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be variously prepared depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. when the host is an Escherichia sp., an α-amylase signal sequence, a subtilisin signal sequence, etc. when the host is a Bacillus sp., an MFα signal sequence, a SUC2 signal sequence, etc. when the host is a yeast, and an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, an origin of replication.

본 발명에서 용어, "벡터"는 핵산 서열을 복제할 수 있는 세포로의 도입을 위해서 핵산 서열을 삽입할 수 있는 전달체를 의미한다. 핵산 서열은 외생 (exogenous) 또는 이종 (heterologous)일 수 있다. 벡터로서는 플라스미드, 코스미드 및 바이러스(예를 들면 박테리오파지)를 들 수 있으나, 이에 제한되지 않는다. 당업자는 표준적인 재조합 기술에 의해 벡터를 구축할 수 있다(Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; 및 Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994 등).As used herein, the term "vector" means a carrier into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors by standard recombinant techniques (see, e.g., Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994).

일 구현예에서, 상기 벡터의 제작 시, 상기 항체를 생산하고자 하는 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In one embodiment, when producing the vector, expression control sequences such as promoter, terminator, enhancer, etc., sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be produced.

본 발명에서 용어, "발현 벡터"는 전사되는 유전자 산물 중 적어도 일부분을 코딩하는 핵산 서열을 포함한 벡터를 의미한다. 일부의 경우에는 그 후 RNA 분자가 단백질, 폴리펩타이드, 또는 펩타이드로 번역된다. 발현 벡터에는 다양한 조절서열을 포함할 수 있다. 전사 및 번역을 조절하는 조절서열과 함께 벡터 및 발현 벡터에는 또 다른 기능도 제공하는 핵산 서열도 포함될 수 있다.As used herein, the term "expression vector" means a vector comprising a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, a polypeptide, or a peptide. Expression vectors may contain various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.

본 발명에서 용어, "숙주세포"는 진핵생물 및 원핵생물을 포함하며, 상기 벡터를 복제할 수 있거나 벡터에 의해 코딩되는 유전자를 발현할 수 있는 임의의 형질 전환 가능한 생물을 의미한다. 숙주세포는 상기 벡터에 의해 형질감염(transfected) 또는 형질전환(transformed) 될 수 있으며, 이는 외생의 핵산분자가 숙주세포 내에 전달되거나 도입되는 과정을 의미한다.The term "host cell" in the present invention includes eukaryotes and prokaryotes, and means any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell can be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

일 구현예에서, 상기 숙주 세포는 박테리아 또는 동물세포일 수 있으며, 동물 세포주는 CHO 세포, HEK 세포 또는 NSO 세포일 수 있고, 박테리아는 대장균일 수 있다.In one embodiment, the host cell can be a bacterial or an animal cell, the animal cell line can be a CHO cell, a HEK cell or a NSO cell, and the bacteria can be Escherichia coli.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 또는 이중특이적 또는 다중특이적 항체를 유효성분으로 포함하는 자가면역 질환, 퇴행성신경 질환(neurodegerative disease), 알츠하이머병, 대사 질환(metabolic disease), 심혈관 질환(cardiovascular disease), 죽상동맥경화증(atherosclerosis), 기관 이식 거부반응, 진균, 바이러스, 박테리아 또는 기생충에 의하여 유발되는 질환 또는 증상으로 이루어진 군으로부터 선택되는 질병 또는 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease or condition selected from the group consisting of autoimmune diseases, neurodegenerative diseases, Alzheimer's disease, metabolic diseases, cardiovascular diseases, atherosclerosis, organ transplant rejection, diseases or symptoms caused by fungi, viruses, bacteria or parasites, comprising an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, or a bispecific or multispecific antibody of the present invention as an active ingredient.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 항원 결합 도메인으로 포함하는 키메라 항원 수용체(Chimeric antigen receptor: CAR)에 관한 것이다.In one aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an antibody of the present invention or a fragment having immunological activity thereof as an antigen binding domain.

일 측면에서, 본 발명은 상기 키메라 항원 수용체를 암호화하는 유전자를 포함하는 재조합 벡터에 관한 것이다.In one aspect, the present invention relates to a recombinant vector comprising a gene encoding the chimeric antigen receptor.

일 측면에서, 본 발명은 상기 재조합 벡터로 형질전환된 키메라 항원 수용체 발현 세포에 관한 것이다.In one aspect, the present invention relates to a chimeric antigen receptor expressing cell transformed with the recombinant vector.

일 구현예에서, 키메라 항원 수용체 발현 세포는 키메라 항원 수용체 발현 대식세포(CAR-macrophage), 키메라 항원 수용체 발현 T(CAR-T) 세포, 키메라 항원 수용체 발현-감마-델타 T(CAR-Gamma-delta T) 세포 또는 자연살해(CAR-NK) 세포일 수 있다.In one embodiment, the chimeric antigen receptor expressing cell can be a chimeric antigen receptor expressing macrophage (CAR-macrophage), a chimeric antigen receptor expressing T (CAR-T) cell, a chimeric antigen receptor expressing-gamma-delta T (CAR-Gamma-delta T) cell, or a natural killer (CAR-NK) cell.

본 발명에서 용어, "CAR(chimeric antigen receptor)"는, 면역 효과기 세포에 특정 항원에 대한 특이성을 부여할 수 있는, 자연적으로 존재하지 않는 수용체를 의미한다. 보통, 상기 CAR은 T 세포에 단일클론항체의 특이성을 이식하기 위하여 사용되는 수용체를 말한다. CAR은 대개 세포 외 도메인(Ectodomain), 막투과성 도메인(transmembrane domain) 및 세포 내 도메인(Ectodomain)으로 구성된다.In the present invention, the term "CAR (chimeric antigen receptor)" refers to a non-naturally occurring receptor capable of imparting specificity for a specific antigen to immune effector cells. Typically, the CAR refers to a receptor used to transplant the specificity of a monoclonal antibody into T cells. CAR is usually composed of an extracellular domain (Ectodomain), a transmembrane domain, and an intracellular domain (Ectodomain).

상기 세포 외 도메인은 항원 결합 부위(antigen recognition region)를 포함하며, CAR의 막통과 도메인은 세포 외 도메인과 연결된 형태로, 자연적 또는 합성된 것에서 유래한 것일 수 있다. 자연적으로 존재하는 것에 유래한 경우, 막 결합 또는 막투과성 단백질에서 유래한 것일 수 있으며, T 세포 수용체의 알파, 베타 또는 제타 체인, CD28, CD3 엡실론, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154 또는 CD8 등 다양한 단백질의 막 투과성 영역에서 유래한 부분일 수 있다. 이러한 막통과 도메인의 서열은 막투과성 단백질의 막투과성 영역 부분을 공지하고 있는 통상의 기술분야에 공지된 문헌 등으로부터 얻을 수 있으나, 이에 제한되는 것은 아니다.The above extracellular domain includes an antigen recognition region, and the transmembrane domain of the CAR is in a form connected to the extracellular domain, and may be derived from natural or synthetic sources. If derived from a naturally existing source, it may be derived from a membrane-bound or membrane-permeable protein, and may be a portion derived from a membrane-permeable region of various proteins such as the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154 or CD8. The sequence of such a transmembrane domain can be obtained from, but is not limited to, documents known in the art that disclose membrane-permeable region portions of membrane-permeable proteins.

또한, 상기 막통과 도메인이 합성된 것일 경우, 이는 류신 및 발린과 같은 소수성 아미노산 잔기를 주로 포함할 수 있으며, 그 예로 페닐알라닌, 트립토판 및 발린의 트리플렛(triplet)이 합성된 막통과 도메인에 존재할 수 있으나, 이에 제한되는 것은 아니다. 이러한 막통과 도메인에 대한 서열정보는 합성된 막통과 도메인에 대한 통상의 기술분야에 공지된 문헌으로부터 얻을 수 있으나, 이에 제한되는 것은 아니다. In addition, when the above-mentioned transmembrane domain is synthetic, it may mainly include hydrophobic amino acid residues such as leucine and valine, and for example, a triplet of phenylalanine, tryptophan and valine may be present in the synthetic transmembrane domain, but is not limited thereto. Sequence information on such transmembrane domain can be obtained from literature known in the art regarding synthetic transmembrane domains, but is not limited thereto.

본 발명의 CAR에서 상기 세포 내 도메인은 세포 내에 존재하는 CAR의 도메인 일부로서, 막통과 도메인과 연결된 형태이다. 본 발명의 상기 세포 내 도메인은 CAR의 항원 결합 부위에 항원이 결합하면 T 세포 활성화, 바람직하게는 T 세포 증식을 가져오는 것이 특징인, 세포 내 신호전달 도메인을 포함할 수 있다. 상기 세포 내 신호전달 도메인은 세포 외에 존재하는 항원 결합 부위에 항체가 결합하면, T 세포 활성화를 가져올 수 있는 신호를 전달하는 부분이라면 특별히 그 종류에 제한되지 않으며, 다양한 종류의 세포 내 신호전달 도메인이 사용될 수 있으며, 그 예로 면역수용체 티로신-기초한 활성화 모티프(tyrosine-based activation motif) 또는 ITAM일 수 있으며, 상기 ITAM은 CD3 제타(ξ, zeta), FcR 감마, FcR 베타, CD3 감마, CD3 델타, CD3 엡실론, CDS, CD22, CD79a, CD79b, CD66d 또는 FcεRIγ에서 유래한 것을 포함하나, 이에 제한되는 것은 아니다.In the CAR of the present invention, the intracellular domain is a part of the domain of the CAR existing within a cell, and is connected to the transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain which is characterized by causing T cell activation, preferably T cell proliferation, when an antigen binds to the antigen binding site of the CAR. The intracellular signaling domain is not particularly limited in its type as long as it is a part that transmits a signal capable of causing T cell activation when an antibody binds to the antigen binding site existing outside the cell, and various types of intracellular signaling domains may be used. An example of this may be an immunoreceptor tyrosine-based activation motif or ITAM, and the ITAM includes, but is not limited to, those derived from CD3 zeta (ξ, zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD66d or FcεRIγ.

CAR는 T-세포 신호화 분자의 세포질 도메인에 경첩 및 막관통 영역을 통해 커플링되는 종양 연계 항원 (TAA)에 특이적인 항체의 단쇄 단편 가변부 (scFv)를 포함한다. 대부분의 통상적인 림프구 활성화 모이어티는 T-세포 촉발 (예를 들어 CD3ζ) 모이어티를 갖는 탠덤(tandem)에서 T-세포 공자극 (예를 들어 CD28, CD137, OX40, ICOS, 및 CD27) 도메인을 포함한다. CAR-매개 입양 면역요법은 CAR-이식된 세포가 비-HLA-제한 방식으로 표적 종양 세포 상의 TAA를 직접 인식하게 한다.CARs comprise a single-chain fragment variable region (scFv) of an antibody specific for a tumor-associated antigen (TAA) that is coupled via the hinge and transmembrane regions to the cytoplasmic domain of a T-cell signaling molecule. Most common lymphocyte activating moieties comprise a T-cell costimulatory (e.g., CD28, CD137, OX40, ICOS, and CD27) domain in tandem with a T-cell triggering (e.g., CD3ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-engrafted cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.

본 발명에서 용어, "키메라 항원 수용체 발현 T(CAR-T) 세포"는 CAR을 발현하는 T 세포를 의미한다. 상기 키메라 항원 수용체 발현 T(CAR-T) 세포는 ⅰ) HLA(human leukocyte antigen)에 비의존적인 방식으로 암 항원을 인지하므로, 세포 표면에 HLA 발현을 감소시켜 항암제의 작용을 회피하는 암들을 치료할 수 있는 이점을 지니며, ⅱ) HLA 타입과 무관하므로, 환자의 HLA 타입과 관계없이 치료에 이용할 수 있으며, ⅲ) 짧은 시간 내에 많은 양의 암 특이적 T 세포를 만들어 낼 수 있으므로, 우수한 항암 효과를 나타낼 수 있는 이점을 지닌다.In the present invention, the term "chimeric antigen receptor expressing T (CAR-T) cell" refers to a T cell expressing a CAR. The chimeric antigen receptor expressing T (CAR-T) cell has the advantage of: i) recognizing a cancer antigen in a manner independent of HLA (human leukocyte antigen), and thus capable of treating cancers that evade the action of anticancer agents by reducing HLA expression on the cell surface; ii) being independent of the HLA type, and thus capable of being used for treatment regardless of the patient's HLA type; and iii) capable of producing a large amount of cancer-specific T cells in a short period of time, and thus capable of exhibiting an excellent anticancer effect.

상기 T 세포는 CD4+ T세포(도움 T 세포, TH 세포), CD8+ T세포(세포독성 T 세포, CTL), 기억 T 세포, 조절 T 세포(Treg 세포) 자연살해 T 세포 등 있으며, 본 발명에서 CAR이 도입되는 T 세포는 바람직하게는 CD8+ T세포 이나, 이에 제한되지 않는다.The above T cells include CD4 + T cells (helper T cells, TH cells), CD8 + T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), natural killer T cells, etc., and the T cells into which the CAR is introduced in the present invention are preferably CD8 + T cells, but are not limited thereto.

일 구현예에서, 본 발명은 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 T 세포 관여자(T-cell engager)에 관한 것이다.In one embodiment, the invention relates to a T-cell engager comprising an antibody or a fragment having immunological activity thereof.

일 구현예에서, 상기 T 세포 관여자는 예를 들어, 이중-특이적 T-세포 관여자 (bispecific T-cell engager, BiTE)일 수 있다. 상기 BiTE는 인공 이중특이적 모노클로날 항체의 부류로, 약 55 킬로달톤의 단일 펩티드 사슬 상에 4 개의 상이한 유전자로부터 아미노산 서열 또는 상이한 항체의 2 개의 단쇄 가변부 단편(scFv) 으로 구성되는 융합 단백질이다. scFv 중 하나는 CD3 수용체를 통해 T 세포에 결합하고, 다른 하나는 종양 특이적인 분자를 통해 종양 세포에 결합한다. 다른 이중특이적 항체와 유사하게, 그리고 보통의 모노클로날 항체와는 달리, BiTE는 T 세포와 종양 세포 사이에서 연결을 형성한다. 이는 퍼포린 및 그랜자임과 같은 단백질을 생산함으로써 MHC I 또는 공자극 분자의 존재와는 독립적으로 T 세포가 종양 세포 상에서 세포독성 활성을 발휘하게 한다. 이들 단백질은 종양 세포로 진입하고 세포의 세포사멸을 개시한다.In one embodiment, the T cell engager can be, for example, a bispecific T-cell engager (BiTE). The BiTE is a class of artificial bispecific monoclonal antibodies, which are fusion proteins consisting of amino acid sequences from four different genes or two single chain variable region fragments (scFv) of different antibodies on a single peptide chain of about 55 kilodaltons. One of the scFv binds to the T cell via the CD3 receptor, and the other binds to the tumor cell via a tumor-specific molecule. Similar to other bispecific antibodies, and unlike conventional monoclonal antibodies, the BiTE forms a link between the T cell and the tumor cell. It causes the T cell to exert cytotoxic activity on the tumor cell independently of the presence of MHC I or costimulatory molecules by producing proteins such as perforin and granzymes. These proteins enter the tumor cell and initiate apoptosis of the cell.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체, 키메라 항원 수용체 발현 세포 또는 T 세포 관여자를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an antibody of the present invention or a fragment thereof having immunological activity, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell or a T cell engager.

일 구현예에서, 암은 뇌종양, 흑색종, 골수종, 비소세포성폐암, 구강암, 간암, 위암, 결장암, 유방암, 삼중음성유방암(Triple Negative Breast Cancer, TNBC), 폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 나팔관암종, 항문부근암, 자궁내막암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으며, 면역관문 억제제에 대해 불응성 암일 수 있다.In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem It may be any one selected from the group consisting of glioma and pituitary adenoma, and may be a cancer refractory to immune checkpoint inhibitors.

본 발명의 약학적 조성물은 단독의 요법으로 이용될 수 있으나, 다른 통상적인 생물학적 요법, 화학 요법 또는 방사 요법과 함께 이용될 수도 있으며, 이러한 병행 요법을 실시하는 경우에는 보다 효과적으로 암을 치료할 수 있다. 본 발명을 암의 예방 및 치료에 이용하는 경우 상기 조성물과 함께 이용될 수 있는 화학 요법제는 시스플라틴(cisplatin), 카르보플라틴(carboplatin), 프로카르바진(procarbazine), 메클로레타민(mechlorethamine), 시클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 멜팔란(melphalan), 클로라부실(chlorambucil), 비술판(bisulfan), 니트로소우레아(nitrosourea), 디악티노마이신(dactinomycin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 블레오마이신(bleomycin), 플리코마이신(plicomycin), 미토마이신(mitomycin), 에토포시드(etoposide), 탁목시펜(tamoxifen), 택솔(taxol), 트랜스플라티눔(transplatinum), 5-플루오로우라실(5-fluorouracil), 빈크리스틴(vincristin), 빈블라스틴(vinblastin) 및 메토트렉세이트(methotrexate) 등을 포함한다. 본 발명의 조성물과 함께 이용될 수 있는 방사 요법은 X-선 조사 및 γ-선 조사 등이다.The pharmaceutical composition of the present invention can be used as a single therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy, and when such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapeutic agents that can be used together with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, etc. Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation, etc.

일 구현예에서, 본 발명의 조성물은 면역원성 세포사멸 유도제를 추가로 포함할 수 있으며, 면역원성 세포사멸 유도제는 안트라사이클린계열 항암제, 탁산 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside), 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제, LV-tSMAC, Measles 바이러스, 블레오마이신(bleomycin), 미토잔트론(mitoxantrone) 또는 옥살리플라틴(oxaliplatin)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으며, 안트라사이클린 계열 항암제는 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin) 또는 발루비신(valrubicin)일 수 있고, 탁산계열 항암제는 파클리탁셀(paclitaxel) 또는 도세탁셀(docetaxel)일 수 있다.In one embodiment, the composition of the present invention may further comprise an immunogenic apoptosis inducer, wherein the immunogenic apoptosis inducer may be at least one selected from the group consisting of an anthracycline series anticancer agent, a taxane series anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib, a cardiac glycoside, a cyclophosphamide series anticancer agent, a GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone, or oxaliplatin, and the anthracycline series anticancer agent may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or It could be valrubicin, and the taxane family of chemotherapy agents could be paclitaxel or docetaxel.

본 발명의 암의 예방 또는 치료용 약학적 조성물은 화학적 항암 약물(항암제) 등과 함께 투여함으로써, 암세포의 사멸 효과를 통해 종래의 항암제의 암치료 효과를 증가시킬 수 있다. 병용 투여는 상기 항암제와 동시에 또는 순차적으로 이루어질 수 있다. 상기 항암제의 예시에는 DNA 알킬화제(DNA alkylating agents)로 메클로에타민(mechloethamine), 클로람부칠(chlorambucil), 페닐알라닌(phenylalanine), 무스타드(mustard), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 카르무스틴(carmustine: BCNU), 로무스틴(lomustine: CCNU), 스트렙토조토신(streptozotocin), 부술판(busulfan), 티오테파(thiotepa), 시스플라틴(cisplatin) 및 카보플라틴(carboplatin); 항암 항생제(anti-cancer antibiotics)로 닥티노마이신(dactinomycin: actinomycin D), 플리카마이신(plicamycin) 및 마이토마이신 C(mitomycin C); 및 식물 알카로이드(plant alkaloids)로 빈크리스틴(vincristine), 빈블라스틴(vinblastine), 에토포시드(etoposide), 테니포시드(teniposide), 토포테칸(topotecan) 및 이리도테칸(iridotecan) 등이 포함되지만, 이에 한정되는 것은 아니다.The pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the cancer cell killing effect by being administered together with a chemical anticancer drug (anticancer agent), etc. The combined administration can be performed simultaneously with or sequentially with the anticancer agent. Examples of the anticancer agent include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; Anti-cancer antibiotics include, but are not limited to, dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan.

본 발명에서, 용어 "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 암의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of cancer by administering a pharmaceutical composition according to the present invention.

본 발명에서 사용되는 용어 "치료"란 본 발명의 조성물의 투여로 암세포의 사멸 또는 암의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.The term "treatment" used in the present invention means all acts that kill cancer cells or improve or beneficially change the symptoms of cancer by administering the composition of the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs will be able to know the exact criteria for diseases to which the composition of the present invention is effective and determine the degree of improvement, enhancement, and treatment by referring to materials presented by the Korean Medical Association, etc.

본 발명에서 유효성분과 결합하여 사용된 "치료학적으로 유효한 양"이란 용어는 대상 질환을 예방 또는 치료하는데 유효한 조성물의 약학적으로 허용가능한 염의 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어 있다.The term "therapeutically effective amount" used in combination with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of the composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, the target site, the condition of the patient, etc. Therefore, the dosage when used in humans should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. Such considerations when determining the effective amount are described in, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.

본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 암의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used in the present invention means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects, and the effective dosage level can be determined according to factors including the patient's health condition, the type and severity of cancer, the activity of the drug, the sensitivity to the drug, the administration method, the administration time, the administration route and the excretion rate, the treatment period, the drug used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

본 발명의 약학적 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may also include a carrier, a diluent, an excipient or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, a diluent, a dispersant, a surfactant, a binder and a lubricant may be additionally added to formulate the composition into a main-use dosage form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 주사 제형으로 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.0001 ~ 10 ㎎/㎖이며, 바람직하게는 0.0001 ~ 5 ㎎/㎖이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The composition of the present invention can be administered non-orally (for example, intravenously, subcutaneously, intraperitoneally, or locally as an injection formulation) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer once a day or in divided doses several times.

본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid preparations for oral administration of the composition of the present invention include suspensions, solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자를 유효성분으로 포함하는 항암 보조제에 관한 것이다.In one aspect, the present invention relates to an anticancer adjuvant comprising as an active ingredient an antibody of the present invention or a fragment having immunological activity thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager.

일 구현예에서, 본 발명의 항암 보조제는 면역항암제와 동시에, 별도로, 또는 순차적으로 병용 투여될 수 있다.In one embodiment, the anticancer adjuvant of the present invention may be administered concurrently, separately, or sequentially with an immunotherapy agent.

일 구현예에서, 본 발명의 항암 보조제는 면역항암제에 대한 불응성을 개선킬 수 있다.In one embodiment, the anticancer adjuvant of the present invention can improve responsivity to immunotherapy.

일 구현예에서, 면역항암제는 면역관문 억제제(Immune checkpoint inhibitors), 면역억제인자 제어 약물, 암 백신, 면역아주번트(immunoadjuvant), 암 치료용 면역세포, 면역세포 활성 보조인자, 암 치료용 항체 또는 암 치료용 면역세포의 활성 유지에 필요한 사이토카인일 수 있다.In one embodiment, the immunotherapy agent may be an immune checkpoint inhibitor, an immunosuppressant modulating drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine required for maintaining the activity of an immune cell for cancer treatment.

일 구현예에서, 면역억제인자 제어 약물은 조절 T 세포(regulatory T cells, Treg)의 수준을 감소시키는 약물일 수 있다.In one embodiment, the immunosuppressant controlling agent may be a drug that reduces the level of regulatory T cells (Treg).

일 구현예에서, 면역관문 억제제는 CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA 또는 A2aR의 억제제일 수 있다.In one embodiment, the immune checkpoint inhibitor can be an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체, 키메라 항원 수용체 발현 세포 또는 T 세포 관여자를 유효성분으로 포함하는 암 진단용 조성물에 관한 것이다.In one aspect, the present invention relates to a composition for diagnosing cancer, comprising as an active ingredient an antibody of the present invention or a fragment thereof having immunological activity, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor expressing cell, or a T cell engager.

일 구현예에서, 표지는 발색효소, 방사성 동위원소, 크로모포어(chromopore), 발광물질, 형광물질, 프로브 또는 택(tag)일 수 있으며, 형광물질은 Cy(cyanine) 계열, 로다민(Rhodamine) 계열, 알렉사(Alexa) 계열, BODIPY 계열 또는 ROX 계열의 형광물질일 수 있고, 나일 레드 (Nile Red), 보디피 (BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), 시아닌 (cyanine), 플루오레세인 (fluorescein), 로다민 (rhodamine), 쿠마린 (coumarine) 또는 알렉사 (Alexa)일 수 있다.In one embodiment, the label can be a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe or a tag, and the fluorescent substance can be a fluorescent substance of the Cy(cyanine) series, Rhodamine series, Alexa series, BODIPY series or ROX series, and can be Nile Red, BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine or Alexa.

일 측면에서, 본 발명은 a) 본 발명의 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자를 포함하는 벡터로 형질전환된 숙주 세포를 배양하는 단계; 및 b) 숙주세포에 의해 발현된 폴리펩타이드를 회수하는 단계를 포함하는, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편의 제조방법에 관한 것이다.In one aspect, the present invention relates to a method for producing an antibody or an immunologically active fragment thereof specific for EphA10, comprising the steps of: a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof; and b) recovering the polypeptide expressed by the host cell.

일 측면에서, 본 발명은 a) 본 발명의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자를 포함하는 벡터로 형질전환된 숙주 세포를 배양하는 단계; 및 b) 숙주세포에 의해 발현된 폴리펩타이드를 회수하는 단계를 포함하는, 이중특이적 또는 다중특이적 항체의 제조방법에 관한 것이다.In one aspect, the present invention relates to a process for producing a bispecific or multispecific antibody, comprising the steps of: a) culturing a host cell transformed with a vector comprising an isolated nucleic acid molecule encoding a bispecific or multispecific antibody of the present invention; and b) recovering the polypeptide expressed by the host cell.

일 구현예에서, 항체의 정제는 여과, HPLC, 음이온 교환 또는 양이온 교환, 고속 액체 크로마토그래피(HPLC), 친화도 크로마토그래피, 또는 이들의 조합을 하는 것이 포함될 수 있으며, 바람직하게는 Protein A를 사용하는 친화 크로마토그래피를 이용할 수 있다.In one embodiment, purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using Protein A.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자의 암의 예방 또는 치료 용도에 관한 것이다.In one aspect, the present invention relates to the use of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention for the prevention or treatment of cancer.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자를 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암의 치료 방법에 관한 것이다.In one aspect, the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention.

일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편, 항체-약물 접합체, 이중특이적 또는 다중특이적 항체, 키메라 항원 수용체 발현 세포, 또는 T 세포 관여자의 암 진단 용도에 관한 것이다.In one aspect, the present invention relates to the use of an antibody or an immunologically active fragment thereof, an antibody-drug conjugate, a bispecific or multispecific antibody, a chimeric antigen receptor expressing cell, or a T cell engager of the present invention for the diagnosis of cancer.

하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only intended to concretize the content of the present invention and the present invention is not limited thereto.

실시예 1. EphA10 항원 단백질의 발현 및 정제Example 1. Expression and purification of EphA10 antigen protein

면역관문 억제인자를 표적하는 치료제에 대한 불응성 유발 인자인 세포막 단백질인 EphA10(Eph receptor A10)을 표적하는 항체 제작용 항원을 제작하기 위해, EphA10의 세포 외부 영역(Extra Cellular Domain, ECD) 또는 세포 외부 영역의 FNIII 도메인 발현용 벡터를 제작하고 이를 동물세포에서 발현 및 정제하였다. 이를 위해, EphA10의 ECD 또는 FNIII 도메인 동물세포 발현용 벡터를 제작하고, 결합활성(Avidity)에 의한 보다 효과적인 항체 스크리닝을 위해, GST(Glutathione-S-transferase) 또는 human Fc가 각각 융합(fusion)된 이합체(dimeric) 형태의 EphA10 단백질 동물세포 발현용 벡터와, 스트렙타비딘(Streptavidin)이 융합된 사합체(tetrameric) 형태의 EphA10 단백질 발현용 동물세포 발현용 벡터도 제작하였다. 구체적으로, Expi293F 세포를 2 × 106 cells/ml의 밀도로 계대배양하고 하루 뒤에, PEI(Polyethylenimine, Polyscience, 23966)를 이용하여 상기에서 제작한 벡터들을 각각 트랜스펙션(transfection)하였다. CO2 인큐베이터에서 37 ℃, 125 rpm 및 8%의 CO2 조건으로 7일간 진탕 배양한 뒤, 원심분리하여 상등액을 취하고, 상등액 부피의 1/25의 25× PBS를 첨가한 뒤 0.2 μm 바틀탑 필터 (Merck Millipore)를 이용하여 여과하였다. 여과된 용액에 GSH 레진, Protein A 레진 또는 Ni-NTA 레진 1 ml에 각각 첨가하고, 4 ℃에서 16 시간 동안 교반한 뒤, 컬럼에 흘려주어 각 레진을 회수하였다. 5 ml 1× PBS로 세척한 후 4 ml의 10 mM 환원된 글루타치온(reduced glutathione)을 포함하는 50 mM Tris-HCl (pH 8.0), 200 mM 글라이신 (pH 2.7) 또는 250 mM 이미다졸(imidazole)로 각각 용출(elution)하였다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 1× PBS로 버퍼 교체한 후 SDS-PAGE를 통해 정제된 10종의 항원 단백질들의 크기와 순도를 분석하였다 (도 1). In order to produce an antigen for producing an antibody targeting EphA10 (Eph receptor A10), a cell membrane protein that is a factor causing nonresponsiveness to therapeutics targeting immune checkpoint inhibitors, a vector for expressing the extra cellular domain (ECD) of EphA10 or the FNIII domain of the extra cellular domain was produced, and it was expressed and purified in animal cells. To this end, an animal cell expression vector for the ECD or FNIII domain of EphA10 was produced, and for more effective antibody screening by binding activity (avidity), an animal cell expression vector for a dimeric EphA10 protein fused with GST (Glutathione-S-transferase) or human Fc, respectively, and an animal cell expression vector for expressing a tetrameric EphA10 protein fused with streptavidin were also produced. Specifically, Expi293F cells were sub-cultured at a density of 2 × 10 6 cells/ml, and one day later, each vector constructed above was transfected using PEI (Polyethylenimine, Polyscience, 23966). After shaking culture for 7 days under the conditions of 37 ℃, 125 rpm, and 8% CO 2 in a CO 2 incubator, centrifugation was performed, and 25× PBS (1/25 of the supernatant volume) was added, followed by filtering using a 0.2 μm bottle-top filter (Merck Millipore). 1 ml of GSH resin, Protein A resin, or Ni-NTA resin was added to the filtered solution, stirred at 4 ℃ for 16 hours, and then flowed through a column to recover each resin. After washing with 5 ml of 1× PBS, the samples were eluted with 50 mM Tris-HCl (pH 8.0), 200 mM glycine (pH 2.7), or 250 mM imidazole containing 4 ml of 10 mM reduced glutathione, respectively. After buffer exchange with 1× PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of the 10 purified antigen proteins were analyzed by SDS-PAGE (Fig. 1).

실시예 2. EphA10 결합 마우스 항체 확보 및 인간화Example 2. Acquisition and humanization of EphA10-binding mouse antibodies

실시예 1에서 제작한 10종의 항원을 이용하여 마우스 면역화 및 하이브리도마 기술 Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495-497)로 EphA10 특이적 마우스 항체 m#2를 확보하였고 이를 바탕으로 인간 불변 영역 (constant region)이 도입된 Chimeric #2 항체를 동물세포 발현 및 항체 정제를 통해 확보하였다 (도 2). 제작한 EphA10 특이적 마우스 항체의 프레임워크 영역(framework region)의 서열을 인간화하여 면역원성(immunogenicity) 유발 가능성을 최소화하기 위해, EphA10 특이적 마우스 항체의 생식세포계열(germline) 서열 분석을 진행하였으며, 이를 통해, EphA10 특이적 마우스 항체와 유사도가 가장 높은 각각 2가지의 인간 중쇄 (IGHV2-5*09, IGHV4-38-2*02) 및 경쇄 (IGKV2-30*02, IGKV2-29*02)의 프레임워크들을 각각 도입한 EphA10 특이적 인간화 항체 4종 (1-1, 1-2, 2-1 및 2-2)을 확보하였다. 또한 현재 임상에 널리 사용되고 있는 치료용 항체들 중, 물성이 매우 우수한 인간화 항체인 트라스트주맙(trastuzumab)의 프레임워크를 도입한 1종의 인간화 항체 (TRA #2)도 추가로 제조하였다 (도 3). Using the 10 antigens produced in Example 1, EphA10-specific mouse antibody m#2 was secured through mouse immunization and hybridoma technology (Kohler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256:495-497), and based on this, chimeric #2 antibody with human constant region introduced was secured through animal cell expression and antibody purification (Fig. 2). In order to minimize the possibility of inducing immunogenicity by humanizing the sequence of the framework region of the produced EphA10-specific mouse antibody, germline sequence analysis of the EphA10-specific mouse antibody was performed, and through this, four types of EphA10-specific humanized antibodies (1-1, 1-2, 2-1, and 2-2) were secured by introducing the frameworks of two human heavy chains (IGHV2-5*09, IGHV4-38-2*02) and light chains (IGKV2-30*02, IGKV2-29*02) that have the highest similarity to the EphA10-specific mouse antibody, respectively. In addition, one humanized antibody (TRA #2) that introduced the framework of trastuzumab, a humanized antibody with excellent physical properties among therapeutic antibodies currently widely used in clinical practice, was also additionally produced (Fig. 3).

실시예 3.Example 3. EphA10 결합 인간화 항체의 발현 및 정제Expression and purification of EphA10-binding humanized antibodies

상기 실시예 2에서 확보한 5종의 EphA10 특이적 인간화 항체들을 IgG 형태로 발현하고 EphA10과의 결합력이 유지되는지 분석하기 위해. 중쇄 및 경쇄 발현 벡터를 각각 3종씩 제작하였다. Expi293F 세포를 2 × 106 cells/ml의 밀도로 계대배양하고 하루 뒤에, PEI(Polyethylenimine, Polyscience, 23966)에 상기에서 제작한 중쇄 발현 벡터 및 경쇄 발현 벡터를 첨가하여 트랜스펙션한 후, CO2 인큐베이터에서 37 ℃, 125 rpm 및 8%의 CO2 조건으로 7일간 진탕 배양하였다. 배양 후 원심분리하여 상등액을 취하고, 상등액 부피의 1/25의 25× PBS를 첨가한 뒤 0.2 μm 바틀탑 필터 (Merck Millipore)를 이용하여 여과하였다. 여과된 용액에 500 μl의 Protein A resin을 첨가하여 4 ℃에서 16 시간 동안 교반한 뒤, 컬럼에 흘려주어 레진을 회수하였다. 5 ml 1× PBS로 세척한 후 3 ml의 100 mM 글라이신 (pH 2.7)로 용출하고 1 M Tris-HCl (pH 8.0)을 이용하여 중화시켰다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 1× PBS로 버퍼 교체한 후 SDS-PAGE를 통해 정제된 5종 항체의 크기와 순도를 분석하였다 (도 4).In order to express the five EphA10-specific humanized antibodies obtained in Example 2 in the form of IgG and to analyze whether the binding affinity to EphA10 is maintained, three types of heavy and light chain expression vectors were produced. Expi293F cells were sub-cultured at a density of 2 × 10 6 cells/ml, and one day later, transfected with the heavy and light chain expression vectors produced above into PEI (Polyethylenimine, Polyscience, 23966), and then cultured with shaking in a CO 2 incubator at 37 °C, 125 rpm, and 8% CO 2 for 7 days. After culture, the supernatant was centrifuged, and 25× PBS (1/25 of the supernatant volume) was added, and then filtered using a 0.2 μm bottle-top filter (Merck Millipore). 500 μl of Protein A resin was added to the filtered solution, stirred at 4 °C for 16 h, and then flowed through the column to recover the resin. After washing with 5 ml of 1× PBS, the solution was eluted with 3 ml of 100 mM glycine (pH 2.7) and neutralized with 1 M Tris-HCl (pH 8.0). After replacing the buffer with 1× PBS using Centrifugal filter units 3K (Merck Millipore), the size and purity of the five purified antibodies were analyzed through SDS-PAGE (Fig. 4).

실시예 4.Example 4. EphA10 특이적 인간화 항체 선별Screening of EphA10-specific humanized antibodies

4-1. EphA10에 대한 결합력 분석4-1. Binding analysis for EphA10

상기 실시예 3에서 정제한 5종의 EphA10 결합 인간화 항체의 EphA10에 대한 결합력을 ELISA로 분석하였다. 구체적으로, human Fc를 융합한 EphA10 4 μg/ml를 0.05 M Na2CO3 (pH 9.6) 용액으로 희석하여 50 μl씩 Flat Bottom Polystyrene High Bind 96 웰 마이크로플레이트 (costar)에 4℃에서 16시간 동안 고정화한 후, 100 μl의 4% 스킴밀크 (GenomicBase)로 상온에서 2 시간 동안 블로킹하였다. 0.05% Tween20가 포함된 1× PBS (PBST) 180 μl로 4 회씩 세척한 뒤, 1% 스킴밀크가 포함된 PBS로 연속 희석된 5종의 항체 (IMGT 1-1, IMGT 1-2, IMGT 2-1, IMGT 2-2 및 TRA #2)를 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 세척 후 goat 항-human kappa chain HRP conjugate (Millipore) 50 μl씩을 첨가하여 상온에서 1 시간 동안 인큐베이션하고, 세척하였다. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific)을 50 μl씩 첨가하여 발색한 뒤 2 M의 H2SO4를 50 μl씩 넣어 반응을 종료시킨 후 Epoch Microplate Spectrophotometer (BioTek)를 이용하여 분석하였다. 그 결과, 인간화 항체들은 인간화 과정 전의 마우스 항체 (ch#2)에 비해 EphA10에 대한 결합력이 크게 감소하지 않고 성공적으로 인간화가 진행된 것을 확인하였다 (도 5). The binding ability of the five kinds of EphA10-binding humanized antibodies purified in Example 3 to EphA10 was analyzed by ELISA. Specifically, 4 μg/ml of EphA10 fused to human Fc was diluted with 0.05 M Na 2 CO 3 (pH 9.6) solution, 50 μl each was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) at room temperature for 2 hours. After washing four times with 180 μl of 1× PBS (PBST) containing 0.05% Tween 20, 50 μl of five types of antibodies (IMGT 1-1, IMGT 1-2, IMGT 2-1, IMGT 2-2, and TRA #2) serially diluted in PBS containing 1% skim milk were dispensed into each well and reacted for 1 h at room temperature. After washing, 50 μl of goat anti-human kappa chain HRP conjugate (Millipore) was added, incubated for 1 h at room temperature, and washed. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added (50 μl) to develop color, and the reaction was terminated by adding 50 μl of 2 M H 2 SO 4. The mixture was then analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, it was confirmed that the humanized antibodies were successfully humanized without a significant decrease in binding affinity to EphA10 compared to the mouse antibody (ch#2) before the humanization process (Fig. 5).

4-2. 세포-발현-EphA10 대한 결합력 분석 및 항체 선별4-2. Binding analysis and antibody selection for cell-expressed EphA10

상기 실시예 3에서 정제한 5종의 EphA10 결합 인간화 항체의 세포-발현-EphA10 대한 결합력을 분석하고, 강한 결합력을 가지는 항체를 선별하였다. 구체적으로, MDA-MB-231 인간 EphA10 과발현 세포주 및 마우스 EphA10 과발현 세포주를 DPBS (Corning)로 현탁하고 세포수가 9 × 104 개가 되도록 각 e-tube에 100 ul씩 분주하였다. 1st 항체로, 상기 실시예 3에서 정제한 5종의 EphA10 결합 인간화 항체 5종과 대조군으로 인간화 과정 전의 마우스 항체 (Chimeric #2) 및 IgG를 500 ng씩 처리하여 4 ℃에서 30분 동안 인큐베이션하였으며, 2nd 항체로 Alexa Fluor 488 anti-human IgG (Invitrogen)을 1:100의 비율로 처리한 뒤 차광하여 4 ℃에서 30분 동안 인큐베이션하였다. DPBS로 2회 세척한 후 CytoFLEX flow cytometer (Beckman Coulter)로 분석하였다. 그 결과, 인간화 과정 전의 마우스 항체 (Chimeric #2)와 5개의 인간화 항체 모두 인간과 마우스 EphA10에 특이적으로 결합했으며, 모든 인간화 항체가 마우스 EphA10에 비해 인간 EphA10에 더 강하게 결합하는 것으로 나타났다. 또한, 5개의 인간화 항체 중 1-1 및 1-2가 EphA10에 가장 강력한 결합력을 보여 최종 후보 대상으로 선정하였다 (도 6).The binding affinity of the five EphA10-binding humanized antibodies purified in Example 3 above to cell-expressed EphA10 was analyzed, and antibodies with strong binding affinity were selected. Specifically, the MDA-MB-231 human EphA10-overexpressing cell line and the mouse EphA10-overexpressing cell line were suspended in DPBS (Corning) and 100 μl was dispensed into each e-tube so that the number of cells was 9 × 10 4 . As a 1st antibody, 5 kinds of EphA10-binding humanized antibodies purified in the above Example 3 and 500 ng of mouse antibody (Chimeric #2) and IgG before humanization as a control were treated and incubated at 4°C for 30 minutes, and as a 2nd antibody, Alexa Fluor 488 anti-human IgG (Invitrogen) was treated at a ratio of 1:100, and then incubated at 4°C for 30 minutes in a light-shielded manner. After washing twice with DPBS, the mixture was analyzed using a CytoFLEX flow cytometer (Beckman Coulter). As a result, both the mouse antibody (Chimeric #2) before humanization and the 5 humanized antibodies specifically bound to human and mouse EphA10, and it was found that all humanized antibodies bound more strongly to human EphA10 than to mouse EphA10. Additionally, among the five humanized antibodies, 1-1 and 1-2 showed the strongest binding affinity to EphA10 and were selected as final candidates (Fig. 6).

실시예 5.Example 5. EphA10 특이적 인간화 항체의 EphA10에 대한 정량적 결합 상수 분석Quantitative binding constant analysis of EphA10-specific humanized antibodies to EphA10

BLI(Bio-Layer Interferometry) 장비인 Octet® BLI system R8 (Satorius)을 이용하여 선별된 2종의 EphA10 특이적 인간화 항체 1-1 및 1-2의 정량적 결합 상수를 분석하였다. 구체적으로, 고정화(Immobilization)를 위해, Octet® Protein A bio-sensor (Satorius)에 5 μg/ml로 200 μl의 1× PBS 에 희석된 2종의 EphA10 특이적 인간화 항체들 (1-1 및 1-2)을 300초 동안 인큐베이션하였다. 1× PBS 를 이용하여 150초 동안 기준선 안정화(baseline stabilization)를 진행한 후에 1,000 nM부터 200 μl의 부피로 1× PBS에 연속 희석(serially dilution)된 EphA10-His 모노머(monomeric) 항원 단백질을 EphA10 특이적 인간화 항체가 코팅된 바이오센서(biosensor)에 300초간 결합(association)시켰다. 그 후, 200 μl의 1× PBS를 300초 동안 인큐베이션하여 해리(dissociation)를 진행하였으며 정량적인 결합 상수들은 Octet BLI Analysis 12.2 software (Satorius)를 이용하여 분석하였다 (도 7).The quantitative binding constants of two kinds of EphA10-specific humanized antibodies 1-1 and 1-2 were analyzed using a BLI (Bio-Layer Interferometry) equipment, Octet ® BLI system R8 (Satorius). Specifically, for immobilization, two kinds of EphA10-specific humanized antibodies (1-1 and 1-2) diluted in 200 μl of 1× PBS at 5 μg/ml were incubated for 300 s on an Octet ® Protein A bio-sensor (Satorius). After baseline stabilization for 150 s using 1× PBS, EphA10-His monomer antigen protein serially diluted in 1× PBS from 1,000 nM to 200 μl in a volume of 1× PBS was associated with the EphA10-specific humanized antibody-coated biosensor for 300 s. Dissociation was then performed by incubating with 200 μl of 1× PBS for 300 s, and quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius) (Fig. 7).

실시예 6.Example 6. EphA10 표적 ADC 항체 제작Production of EphA10-targeting ADC antibodies

상기 실시예 4에서 선별한 EphA10 특이적 인간화 항체 1-1 및 1-2를 이용하여 ADC(Antibody drug conjugate)를 제작하기 위해, 일반적으로 ADC를 제조하는데 사용되고 있는 부분적인 환원에 이은 시스테인 컨쥬게이션(cysteine conjugation) 방식으로 ADC를 제작하였다. 구체적으로, PD MidiTrap G-25 (Cytiva, 28918008)를 사용하여 1× PBS에 보관되었던 EphA10 특이적 인간화 항체 1-1 및 1-2의 버퍼를 환원 버퍼 (5 mM DTPA, pH 8.0 PBS)로 교환한 뒤, Centrifugal filter units 10K (Merck Millipore)을 이용하여 4 mg/ml로 농축하였다. 농축된 항체 2 mg/ml 및 Tris(2-carboxyethyl)phosphine (TCEP, sigma) 68.7 uM가 되도록 환원 버퍼와 혼합하여 2시간 동안 37 ℃에서 환원 반응을 수행하였다. 그 후 TCEP을 제거하여 추가적인 환원을 막고 컨쥬게이션(conjugation)을 위해, 1× PBS 버퍼 (pH 7.4)로 상기의 버퍼 교환과 동일하게 PD MidiTrap G-25 column, Centrifugal filter units 10k를 이용하여 교환 및 농축을 진행하였다. 2 mg/ml 농도의 환원된 항체를 시스테인(cysteine)과 반응하는 링커-약물인 MC-Val-Cit-PAB-MMAE (HY-15575, medchemexpress) 63.2 uM와 1× PBS에서 혼합하여 4 ℃에서 1시간 동안 반응시켰다. 추가 반응을 막기 위해 시스테인을 1× PBS에 녹여, 처리한 링커-약물의 2배 농도인 126.4 μM로 처리하여 10분간 상온에서 퀀칭(quenching)하였다. 반응이 종결된 용액으로 SEC(size exclusion chromatography) 정제를 진행한 뒤, FPLC (Biorad) system에서 Superdex 200 increase 10/300 (28990944, Cytiva) 컬럼에 반응 용액을 주입하고, 0.4 ml/min으로 1× PBS를 이용해 SEC 크로마토그래피를 수행한 후, SDS-PAGE를 통해 분석하였다 (도 8). 용출된 ADC는 Centrifugal filter units 10K로 농축하여 분석과 사용 전까지 -20 ℃에서 보관하였으며, 정제된 ADC는 hydrophobic interaction chromatography 방법으로 분석하였으며, ADC는 DAR에 따라서 발생하는 hydrophobicity 차이에 의해 peak가 분리되었다 (도 8). In order to produce an antibody drug conjugate (ADC) using the EphA10-specific humanized antibodies 1-1 and 1-2 selected in Example 4 above, the ADC was produced by the partial reduction followed by cysteine conjugation method, which is generally used in the production of ADC. Specifically, the buffer of the EphA10-specific humanized antibodies 1-1 and 1-2 stored in 1× PBS was exchanged with a reducing buffer (5 mM DTPA, pH 8.0 PBS) using PD MidiTrap G-25 (Cytiva, 28918008), and then concentrated to 4 mg/ml using Centrifugal filter units 10K (Merck Millipore). The concentrated antibody was mixed with reduction buffer to obtain 2 mg/ml and Tris(2-carboxyethyl)phosphine (TCEP, sigma) 68.7 uM, and reduction reaction was performed at 37°C for 2 hours. TCEP was then removed to prevent further reduction, and exchange and concentration were performed in the same manner as the buffer exchange described above with 1× PBS buffer (pH 7.4) using PD MidiTrap G-25 column, Centrifugal filter units 10k for conjugation. The reduced antibody at a concentration of 2 mg/ml was mixed with 63.2 uM MC-Val-Cit-PAB-MMAE (HY-15575, medchemexpress), a linker drug that reacts with cysteine, in 1× PBS, and reacted at 4°C for 1 hour. To prevent further reaction, cysteine was dissolved in 1× PBS, treated with 126.4 μM, which is twice the concentration of the treated linker-drug, and quenched at room temperature for 10 min. After the reaction was completed, SEC (size exclusion chromatography) purification was performed, and the reaction solution was injected into a Superdex 200 increase 10/300 (28990944, Cytiva) column in an FPLC (Biorad) system, and SEC chromatography was performed using 1× PBS at 0.4 ml/min, and then analyzed by SDS-PAGE (Fig. 8). The eluted ADC was concentrated with Centrifugal filter units 10K and stored at -20 ℃ until analysis and use, and the purified ADC was analyzed by the hydrophobic interaction chromatography method, and the ADC peaks were separated by the difference in hydrophobicity that occurred according to the DAR (Fig. 8).

실시예 7.Example 7. EphA10 표적 ADC 항체의 효능 분석 및 서열 분석Potency analysis and sequence analysis of EphA10-targeting ADC antibodies

7-1. ADC의 암세포 사멸 효과 확인7-1. Confirmation of ADC’s cancer cell killing effect

ADC의 효능을 in vitro로 확인하기 위해, ATP 측정을 통한 세포 생존 분석으로 암세포 사멸 효과를 확인하였다. 구체적으로, MDA-MB-231 naive cell (대조군), mock 세포주 (음성 대조군) 및 hEphA10 과발현 세포주를 각각 96웰 화이트 플레이트 (SPL)에 웰 당 2 × 104 씩 분주하고, 다음 날 상기 실시예 6에서 제작한 ADC를 RPMI (Capricorn)에 농도별로 희석하여 각 웰에 100 μl씩 처리한 후, 37 ℃에서 48시간 동안 인큐베이션하였다. ADC를 처리한 플레이트를 배양기 밖 상온에서 5분간 인큐베이션하고, 각 웰에 CellTiter-Glo 용액을 50 μl씩 첨가한 뒤, 500 rpm에서 2분 동안 shaking하였다. 상온의 실험대에서 10분 동안 차광하여 반응시킨 후 Synergy HTX multimode reader (Biotek)를 이용하여 ATP를 측정하였다. To confirm the efficacy of ADC in vitro , the cancer cell killing effect was confirmed by cell survival analysis through ATP measurement. Specifically, MDA-MB-231 naive cells (control group), mock cell line (negative control group), and hEphA10 overexpressing cell line were each dispensed at 2 × 10 4 per well in a 96-well white plate (SPL), and the following day, the ADC produced in Example 6 was diluted by concentration in RPMI (Capricorn), and 100 μl was treated to each well, followed by incubation at 37 °C for 48 hours. The ADC-treated plate was incubated for 5 minutes at room temperature outside the incubator, 50 μl of CellTiter-Glo solution was added to each well, and then shaken at 500 rpm for 2 minutes. After reacting for 10 minutes on a room temperature bench in a dark place, ATP was measured using a Synergy HTX multimode reader (Biotek).

그 결과, ADC에서 명확한 세포사멸 효과가 관찰되지 않았다 (도 9).As a result, no clear apoptotic effect was observed in ADC (Fig. 9).

7-2. 서열 분석7-2. Sequence analysis

상기 실시예 7-1에서 EphA10 특이적 인간화 항체를 이용한 ADC 항체가 in vitro ADC 효능을 나타내지 않은 이유를 분석하기 위해, EphA10 특이적 인간화 항체의 서열을 다시 분석하였다. 서열 분석 결과, EphA10 특이적 인간화 항체의 경쇄의 CDR 영역에 이황화(disulfide) 결합에 관여하지 않는 자유 시스테인(free cysteine) 아미노산이 존재함을 확인하였다 (도 10). EphA10 표적 ADC 항체는 링커(linker)를 통한 payload의 저분자 물질을 연결하여 생성하였기 때문에 이 과정에서 CDR 영역의 자유 시스테인에 payload가 컨쥬게이션됨으로써 EphA10 표적 인간화 항체의 항원 에피토프(epitope)와 결합하는 부위를 블로킹하여 항원 결합력을 소실하는 것으로 가정하였다. 이를 검증하기 위해 EphA10 특이적 인간화 항체 (1-1)과 ADC 제조후의 EphA10 표적 ADC 항체 (1-1 (ADC))의 EphA10에 대한 결합력을 ELISA로 분석하여 비교한 결과, 예상대로 항체 경쇄의 CDR에 존재하는 자유 시스테인으로 인하여 ADC 항체의 EphA10 결합력이 매우 크게 감소하였음을 확인하였고, 이로 인해 in vitro ADC 효능 또한 크게 감소된다는 사실을 확인하였다 (도 11).In order to analyze the reason why the ADC antibody using the EphA10-specific humanized antibody in the above Example 7-1 did not show in vitro ADC efficacy, the sequence of the EphA10-specific humanized antibody was reanalyzed. The sequence analysis results confirmed that a free cysteine amino acid that is not involved in disulfide bonds exists in the CDR region of the light chain of the EphA10-specific humanized antibody (Fig. 10). Since the EphA10-targeting ADC antibody was produced by linking a low-molecular-weight substance of the payload through a linker, it was assumed that in this process, the payload was conjugated to the free cysteine of the CDR region, thereby blocking the site that binds to the antigen epitope of the EphA10-targeting humanized antibody and thereby losing antigen binding ability. To verify this, the binding affinity of EphA10-specific humanized antibody (1-1) and EphA10-targeting ADC antibody (1-1 (ADC)) after ADC manufacturing was analyzed by ELISA and compared. As a result, it was confirmed that the EphA10 binding affinity of the ADC antibody was greatly reduced due to the free cysteine present in the CDR of the antibody light chain, as expected, and it was confirmed that the in vitro ADC efficacy was also greatly reduced due to this (Fig. 11).

실시예 8. ADC 효능 개선된Example 8. Improved ADC efficacy EphA10 특이적 인간화 항체 제작Production of EphA10-specific humanized antibodies

상기 실시예 7에서 확인한 결과에 따라 실시예 4에서 선별한 EphA10 특이적 인간화 항체 1-1 및 1-2의 경쇄에 존재하는 자유 시스테인을 시스테인과 특성이 유사한 세린(serine) (1-1-S 및 1-2-S) 또는 항체의 전체 구조변화의 영향을 최소화시키기 위한 알라닌(alanine) (1-1-A 및 1-2-A)으로 선택하여 각각 치환하였고, 경쇄 동물세포 발현용 벡터에 각각 클로닝하고, 상기 실시예 3에서와 같이 동물세포에서 각 항체를 발현 및 정제한 뒤, SDS-PAGE를 통해 정제된 항체들 (1-1-A, 1-1-S, 1-2-A 및 1-2-S)의 크기와 순도를 분석하였다 (도 12).According to the results confirmed in the above Example 7, the free cysteine present in the light chain of the EphA10-specific humanized antibodies 1-1 and 1-2 selected in Example 4 was selected and replaced with serine (1-1-S and 1-2-S) which has similar properties to cysteine, or alanine (1-1-A and 1-2-A) to minimize the effect of the overall structural change of the antibody, respectively, and each was cloned into a vector for light chain animal cell expression, and each antibody was expressed and purified in animal cells as in the above Example 3, and the size and purity of the purified antibodies (1-1-A, 1-1-S, 1-2-A, and 1-2-S) were analyzed through SDS-PAGE (Fig. 12).

실시예 9. ADC 효능 개선된Example 9. Improved ADC efficacy EphA10 특이적 인간화 항체의 EphA10에 대한 결합력 분석 Binding Affinity Analysis of EphA10-Specific Humanized Antibodies to EphA10

상기 실시예 8에서 2종의 EphA10 특이적 인간화 항체 1-1 및 1-2의 경쇄에 존재하는 자유 시스테인을 각각 세린 및 알라닌으로 치환한 항체들 (1-1-A, 1-1-S, 1-2-A 및 1-2-S)의 EphA10에 대한 결합력을 ELISA로 분석하였다.In Example 8 above, the binding affinity of antibodies (1-1-A, 1-1-S, 1-2-A, and 1-2-S) in which the free cysteine present in the light chain of two types of EphA10-specific humanized antibodies 1-1 and 1-2 was substituted with serine and alanine, respectively, to EphA10 was analyzed by ELISA.

그 결과, EphA10 특이적 인간화 항체 1-1 및 항체 1-2의 자유 시스테인을 세린으로 치환한 경우 EphA10에 대한 결합력을 거의 완전히 잃어버리는 반면, 알라닌으로 치환한 경우 EphA10에 대한 결합력이 회복되는 것으로 나타났다 (도 13).As a result, when the free cysteine of EphA10-specific humanized antibodies 1-1 and 1-2 was replaced with serine, the binding affinity to EphA10 was almost completely lost, whereas when it was replaced with alanine, the binding affinity to EphA10 was restored (Fig. 13).

실시예 10. EphA10 결합력 향상을 위한 효모 디스플레이 라이브러리 구축 Example 10. Construction of a yeast display library to enhance EphA10 binding affinity

상기 실시예에서 발굴한 EphA10 특이적 인간화 항체들 1-1 및 1-2로부터 EphA10에 대한 결합력이 추가로 향상된 항체들을 발굴하기 위해, 1-1 및 1-2 항체의 가변영역들 (VH 및 VL)에 Error-prone PCR을 통해 0.2%로 무작위적인 돌연변이가 들어가도록 유도하여 증폭한 항체 라이브러리를 구축하였다. 제작된 DNA 라이브러리는 MicroPulser Electroporator (Bio-Rad)를 이용한 전기천공법을 통해 효모종 사카로마이세스 세레비지에(Saccharomyces cerevisiae) 컴피턴트(Competent) 세포 400 μl에 형질전환(transformation)한 뒤, SD 배지 [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD Biosciences) 5.0 g/l, Na2HPO4 (Junsei) 5.4 g/l, NaH2PO4.H2O (Samchun) 8.56 g/l, Glucose (Duksan) 20 g/l] 500 ml에서 배양하여 형질전환된 효모종만 선택적으로 생존하게하여 1-1 및 1-2 각각 7.5 × 107 및 1.0 × 108의 라이브러리 크기를 갖는 효모 디스플레이 라이브러리를 제작하였다 (도 14). In order to discover antibodies with further improved binding affinity to EphA10 from the EphA10-specific humanized antibodies 1-1 and 1-2 discovered in the above examples, an antibody library was constructed by amplifying the variable regions (VH and VL) of antibodies 1-1 and 1-2 by introducing random mutations at 0.2% through error-prone PCR. The constructed DNA library was transformed into 400 μl of competent Saccharomyces cerevisiae cells by electroporation using a MicroPulser Electroporator (Bio-Rad), and then cultured in 500 ml of SD medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD Biosciences) 5.0 g/l, Na 2 HPO 4 (Junsei) 5.4 g/l, NaH 2 PO 4 .H 2 O (Samchun) 8.56 g/l, Glucose (Duksan) 20 g/l] to selectively allow the transformed yeast to survive, resulting in a yeast display library with library sizes of 7.5 × 10 7 and 1.0 × 10 8 for 1-1 and 1-2, respectively. was produced (Fig. 14).

실시예 11. EphA10에 대한 결합력이 향상된Example 11. Enhanced binding affinity to EphA10 인간화 항체 스크리닝 Humanized Antibody Screening

우선, 유세포 분리기를 이용한 스크리닝에 이용하기 위해 human Fc가 융합된 항원 단백질 EphA10-Fc에 Alexa488 형광분자를 표지화하였다. 또한, 상기 실시예 10에서 제작한 효모 라이브러리의 5.5 × 108개 세포를 트립토판(tryptophan)이 결핍된 SD 배지 100 ml에서 30 ℃ 조건으로 16시간 동안 배양한 후, 7 × 108개 세포를 SG 배지 [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid(BD) 5.0 g/l, Na2HPO4(Junsei) 5.4 g/l, NaH2PO4.H2O (SAMCHUN) 8.56 g/l, Galactose (Sigma-Aldrich) 20 g/l] 100 ml에서 20 ℃ 조건으로 48 시간 동안 배양하였다. 배양 세포 중 1 × 108개 세포를 4 ℃에서 14,000 × g로 30초간 원심분리한 후 상층액을 제거하고, 1 ml PBSB (1× PBS, 0.1% bovine serum albumin (Gibco)(pH 7.4)로 세척한 뒤 200 nM의 Alexa488 컨쥬게이트된 EphA10-Fc 및, 1:1,000의 비율로 PBSB (pH 7.4) 1 ml에 희석된 anti-FLAG-iFlour (GenScript)를 첨가하고, 1시간 동안 인큐베이션하였다. 인큐베이션 후 1 ml PBSB (pH 7.4)로 세척한 후에, 1 ml PBSB (pH 7.4)로 재현탁하고, Alexa488 컨쥬게이트된 EphA10-Fc와의 결합을 유도하여 높은 형광 신호를 보이는 세포들을 유세포 분리기 (Bio-Rad S3 sorter, Bio-Rad)를 통해 회수하였다. 회수된 서브-라이브러리(sub-library)는 동일한 방법으로 Alexa488 컨쥬게이트된 EphA10-Fc의 농도를 줄이며 총 5 라운드의 선별 과정을 거쳐 EphA10에 대한 결합력이 우수한 Fab 변이체들을 디스플레이하고 있는 세포들을 농축시켰다 (도 15).First, human Fc-fused antigen protein EphA10-Fc was labeled with Alexa488 fluorescent molecule for screening using a flow cytometer. In addition, 5.5 × 10 8 cells of the yeast library produced in Example 10 were cultured in 100 ml of SD medium lacking tryptophan at 30°C for 16 hours, and then 7 × 10 8 cells were cultured in 100 ml of SG medium [Difco Yeast nitrogen base (BD Biosciences) 6.7 g/l, Bacto casamino acid (BD) 5.0 g/l, Na 2 HPO 4 (Junsei) 5.4 g/l, NaH 2 PO 4 .H 2 O (SAMCHUN) 8.56 g/l, Galactose (Sigma-Aldrich) 20 g/l] at 20°C for 48 hours. Among the cultured cells, 1 × 108 cells were centrifuged at 4 °C, 14,000 × g for 30 s, the supernatant was removed, washed with 1 ml of PBSB (1× PBS, 0.1% bovine serum albumin (Gibco) (pH 7.4), and 200 nM of Alexa488-conjugated EphA10-Fc and anti-FLAG-iFlour (GenScript) diluted 1:1,000 in 1 ml of PBSB (pH 7.4) were added and incubated for 1 hour. After incubation, the cells were washed with 1 ml of PBSB (pH 7.4), resuspended in 1 ml of PBSB (pH 7.4), and the cells showing high fluorescence signals by inducing binding to Alexa488-conjugated EphA10-Fc were separated using a flow cytometer (Bio-Rad S3 The recovered sub-library was collected through a sorter, Bio-Rad. The recovered sub-library was subjected to a total of five rounds of selection using decreasing concentrations of Alexa488-conjugated EphA10-Fc in the same manner to enrich for cells displaying Fab variants with excellent binding affinity to EphA10 (Fig. 15).

실시예 12. EphA10에 대한 결합력이 향상된 인간화 항체의 유전자 서열 확인Example 12. Identification of the genetic sequence of a humanized antibody with enhanced binding affinity to EphA10

상기 실시예 11에서 EphA10에 대한 결합 신호가 우수한 Fab 변이체 클론들의 유전자 서열을 생어 시퀀싱(Sanger sequencing)으로 확인하였다. In Example 11 above, the gene sequences of Fab variant clones with excellent binding signals to EphA10 were confirmed by Sanger sequencing.

분석 결과, 특정 항체 서열을 갖는 항체가 성공적으로 농축(enrich)되었음을 확인하였으며, 상기 EphA10 결합력 향상을 위한 스크리닝을 통해 10종의 항체 (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N 및 2-W)를 확보하였다 (도 16). 또한, 발굴된 항체들의 대부분 클론들은 1-1 항체와 1-2 항체의 경쇄 CDR에 존재하는 자유 시스테인이 아스파라긴(asparagine, N), 아르기닌(arginine, R), 트립토판(tryptophan, W) 또는 티로신(tyrosine, Y) 중의 하나로 치환된 것으로 확인되어, 1-1 항체 및 1-2 항체 각각의 경쇄 CDR에 존재하는 자유 시스테인을 아스파라긴(asparagine), 아르기닌(arginine), 트립토판(tryptophan) 또는 티로신(tyrosine)으로 각각 단일 아미노산 치환 돌연변이 유도한 변이체 6종 (1-R, 1-W, 1-Y, 2-N, 2-R 및 2-Y)을 추가로 제조하였다 (도 16).The analysis results confirmed that antibodies with specific antibody sequences were successfully enriched, and 10 antibodies (A.3, A.7, A.8, B.1, B.3, B.7, 2.1, 2.3, 1-N, and 2-W) were secured through screening for improved EphA10 binding affinity (Fig. 16). In addition, most clones of the discovered antibodies were confirmed to have a free cysteine in the light chain CDR of antibodies 1-1 and 1-2 substituted with one of asparagine (N), arginine (R), tryptophan (W), or tyrosine (Y). Therefore, six mutants (1-R, 1-W, 1-Y, 2-N, 2-R, and 2-Y) were additionally prepared by inducing single amino acid substitution mutations of the free cysteine in the light chain CDR of antibodies 1-1 and 1-2 to asparagine, arginine, tryptophan, or tyrosine, respectively (Fig. 16).

실시예 13. EphA10에 대한 결합력이 향상된 인간화 항체의 발현 및 정제Example 13. Expression and purification of humanized antibodies with enhanced binding affinity to EphA10

상기 실시예 12에서 선별된 16종의 Fab 항체 변이체들이 IgG 형태로도 EphA10과의 결합력을 가지는지를 확인하기 위하여, Fab 항체 변이체들의 중쇄 및 경쇄 발현 벡터들을 각각 제작하고, 실시예 3에서와 같이 동물세포에서 각 항체를 발현 및 정제한 뒤, SDS-PAGE를 통해 각각 정제된 16종 항체들의 크기와 순도를 분석하였다 (도 17).In order to confirm whether the 16 Fab antibody variants selected in Example 12 above also have binding affinity to EphA10 in the form of IgG, expression vectors for the heavy and light chains of the Fab antibody variants were each constructed, and each antibody was expressed and purified in animal cells as in Example 3. Then, the size and purity of each purified 16 antibodies were analyzed through SDS-PAGE (Fig. 17).

실시예 14. EphA10에 대한 결합력이 향상된 인간화 항체의 EphA10 결합력 분석Example 14. Analysis of EphA10 binding affinity of humanized antibodies with enhanced binding affinity to EphA10

상기 실시예 13에서 정제한 16종의 항체들의 EphA10에 대한 결합력이 증가하였는지 확인하기 위하여 ELISA 분석을 수행한 결과, 실시예 10에서 추가 엔지니어링을 통해 선별한 인간화 항체들의 EphA10에 대한 결합력이 향상된 것을 확인하였다 (도 18). In order to confirm whether the binding affinity of the 16 antibodies purified in Example 13 above increased toward EphA10, an ELISA analysis was performed. As a result, it was confirmed that the binding affinity of the humanized antibodies selected through additional engineering in Example 10 improved toward EphA10 (Fig. 18).

실시예 15. EphA10에 대한 결합력이 향상된Example 15. Enhanced binding affinity to EphA10 인간화 항체의 ADC 제작ADC production of humanized antibodies

CDR 영역의 시스테인이 치환되고 추가 엔지니어링을 거쳐 선발된 EphA10 특이적 인간화 항체들 중 2종 (A.7 및 2-W)을 이용하여 실시예 6의 시스테인 컨쥬세이션 방식으로 ADC를 제작하고, SDS-PAGE를 통해 분석하였다 (도 19). 또한, 정제된 ADC는 hydrophobic interaction chromatography 방법으로 분석하였다 (도 19). 제작된 ADC는 DAR에 따라서 발생하는 hydrophobicity 차이에 의해 peak가 분리되었으며, HIC 분석을 통해서 ADC의 DAR(Drug to Antibody ratio) 분포와 그 비율을 확인하였다.Among the EphA10-specific humanized antibodies selected by substituting cysteines in the CDR region and undergoing further engineering, two types (A.7 and 2-W) were used to produce ADCs by the cysteine conjugation method of Example 6, and analyzed by SDS-PAGE (Fig. 19). In addition, the purified ADCs were analyzed by the hydrophobic interaction chromatography method (Fig. 19). The produced ADCs showed peak separation by the difference in hydrophobicity that occurred according to the DAR, and the distribution and ratio of the DAR (Drug to Antibody ratio) of the ADCs were confirmed by HIC analysis.

실시예 16. EphA10에 대한 결합력이 향상된Example 16. Enhanced binding affinity to EphA10 인간화 항체 기반 ADC에 대한 EphA10 항원 결합력 분석 EphA10 antigen binding affinity analysis for humanized antibody-based ADCs

실시예 4에서 선별한 인간화 항체 1-1를 이용하여 제작한 EphA10 표적 ADC (1-1_ADC), 실시예 4에서 선별한 인간화 항체 1-2의 경쇄 CDR에 존재하는 자유 시스테인을 트립토판으로 치환한 항체 (2-W)를 이용하여 제작한 EphA10 표적 ADC (2W_ADC), 및 효모 디스플레이 라이브러리를 통해 선별한 항체 A7 (A.7)를 이용하여 제작한 EphA10 표적 ADC (A7_ADC) 항체들의 EphA10 결합력이 ADC 전과 같이 유지되는지 ELISA로 분석하였다.The EphA10-targeting ADC (1-1_ADC) produced using the humanized antibody 1-1 selected in Example 4, the EphA10-targeting ADC (2W_ADC) produced using an antibody (2-W) in which the free cysteine present in the light chain CDR of the humanized antibody 1-2 selected in Example 4 was substituted with tryptophan, and the EphA10-targeting ADC (A7_ADC) produced using the antibody A7 (A.7) selected through the yeast display library were analyzed by ELISA to see whether the EphA10 binding affinity of the antibodies was maintained at the same level as before the ADC.

그 결과, 실시예 4에서 선별한 인간화 항체 1-1 및 1-2 항체를 ADC로 제조하였을 때 EphA10 결합력이 현저히 감소되었던 것과 달리, 추가적인 엔지니어링 (아미노산 치환, 효모 디스플레이 라이브러리)을 적용하여 선별된 항체로부터 ADC를 제조하였을 때에는 EphA10에 대한 결합력이 매우 잘 유지되는 것으로 확인되었다 (도 20).As a result, unlike the case where the binding affinity to EphA10 was significantly reduced when the humanized antibodies 1-1 and 1-2 selected in Example 4 were produced as ADCs, it was confirmed that the binding affinity to EphA10 was very well maintained when ADCs were produced from the selected antibodies by applying additional engineering (amino acid substitution, yeast display library) (Fig. 20).

실시예 17. EphA10에 대한 결합력이 향상된Example 17. Enhanced binding affinity to EphA10 인간화 항체 기반 ADC의 암세포 사멸 효과 확인Confirmation of cancer cell killing effect of humanized antibody-based ADC

A7 및 2W 항체를 이용하여 제작한 2종의 EphA10 표적 ADC (A7-MMAE 및 2W_MMAE)의 효과를 검증하기 위해 상기 실시예 7-1과 같이 ATP를 측정하여 암세포의 세포 생존율 분석을 수행하였다.To verify the effectiveness of two EphA10 targeting ADCs (A7-MMAE and 2W_MMAE) produced using A7 and 2W antibodies, cell viability analysis of cancer cells was performed by measuring ATP as in Example 7-1.

그 결과, 대조군 항체 (A7 및 W2)는 세포의 생존에 큰 영향을 미치지 않은 반면, 이를 이용한 EphA10 표적 ADC들은 모두 뚜렷하게 암세포 사멸 효과를 나타냈으며, ADC의 암세포 사멸 효과는 EphA10의 발현에 비례하여 증가하는 것으로 나타났다 (도 21).As a result, while the control antibodies (A7 and W2) did not significantly affect cell survival, all EphA10-targeting ADCs using them exhibited a clear cancer cell killing effect, and the cancer cell killing effect of the ADCs was found to increase in proportion to the expression of EphA10 (Fig. 21).

실시예 18. EphA10에 대한 결합력이 향상된Example 18. Enhanced binding affinity to EphA10 인간화 항체의 EphA10에 대한 정량적 결합 상수 분석Quantitative binding constant analysis of humanized antibodies to EphA10

선발된 EphA10 특이적 인간화 항체 2종 (A.7 및 2-W)의 EphA10에 대한 정량적 결합 상수를 분석하기 위해 BLI(Bio-Layer Interferometry) 장비인 Octet® BLI system R8 (Satorius)을 이용하여 실험을 수행하였다. 구체적으로, 고정화(Immobilization)를 위해, Octet® Protein A bio-sensor (Satorius)에 5 μg/ml로 200 μl의 1× PBS 에 희석된 2종의 EphA10 특이적 인간화 항체들 (A.7 및 2-W)을 300초 동안 인큐베이션하였다. 1× PBS 를 이용하여 150초 동안 기준선 안정화를 진행한 후에 1,000 nM부터 200 μl의 부피로 1× PBS에 연속 희석된 EphA10-His 모노머 항원 단백질을 EphA10 특이적 인간화 항체 (A.7 및 2-W)가 각각 코팅된 바이오센서에 300초간 결합시켰다. 그 후, 200 μl의 1× PBS를 300초 동안 인큐베이션하여 해리를 진행하였으며 정량적인 결합 상수들은 Octet BLI Analysis 12.2 software (Satorius)를 이용하여 분석하였다To analyze the quantitative binding constants of two selected EphA10-specific humanized antibodies (A.7 and 2-W) to EphA10, experiments were performed using a bio-layer interferometry (BLI) device, Octet ® BLI system R8 (Satorius). Specifically, for immobilization, two EphA10-specific humanized antibodies (A.7 and 2-W) diluted in 200 μl of 1× PBS at 5 μg/ml were incubated on an Octet ® Protein A bio-sensor (Satorius) for 300 s. After baseline stabilization for 150 s using 1× PBS, serially diluted EphA10-His monomer antigen protein from 1,000 nM to 200 μl in 1× PBS was bound to the biosensor coated with each EphA10-specific humanized antibody (A.7 and 2-W) for 300 s. After that, dissociation was performed by incubating with 200 μl of 1× PBS for 300 s, and quantitative binding constants were analyzed using Octet BLI Analysis 12.2 software (Satorius).

그 결과, 두 항체 모두 #2 항체보다 인간 EphA10에 높은 결합 상수를 가지는 것을 확인하였다 (도 22).As a result, it was confirmed that both antibodies had higher binding constants to human EphA10 than antibody #2 (Fig. 22).

실시예 19. EphA10에 대한 결합력이 향상된Example 19. Enhanced binding affinity to EphA10 인간화 항체의 마우스 EphA10 결합력 분석Analysis of binding affinity of humanized antibodies to mouse EphA10

19-1. 마우스 EphA10의 발현 및 정제19-1. Expression and purification of mouse EphA10

인간 EphA10과 마우스 EphA10은 95%의 유사도를 가지므로, 항체가 마우스 EphA10에 결합한다면 EphA10 표적 항체의 in vivo 실험 전, 마우스 모델 선정에 좋은 지표로 활용 가능하다. 따라서, 마우스 EphA10의 세포 외부 영역(extracellular domain, ECD)을 다양한 융합 단백질과 함께 발현 가능한 동물세포 발현용 벡터를 준비하고, Expi293F 세포에 트랜스펙션하여 7일 동안 배양한 뒤, 원심분리하여 상등액을 취하였다. 상등액의 1/25 부피의 25× PBS를 첨가한 후 0.2 μm bottle top filter (Merck Millipore)를 이용하여 여과하고, 여과된 상등액에 GSH 레진, Protein A 레진 또는 Ni-NTA 레진 1 ml을 첨가한 뒤, 4 ℃에서 16 시간 교반하였다. 이 후 컬럼에 흘려주어 각 레진을 회수하였다. 5 ml 1× PBS로 세척한 후 4 ml의 10 mM 환원된 글루타치온을 포함하는 50 mM Tris-HCl (pH 8.0), 200 mM 글라이신 (pH 2.7) 또는 250 mM 이미다졸로 각각 용출하였다. Centrifugal filter units 3K (Merck Millipore)을 사용하여 1× PBS로 버퍼 교체한 후 SDS-PAGE를 통해 정제된 4종의 항원 단백질들의 크기와 순도를 분석한 결과, 4종의 항원 단백질 중 mEphA10-Fc (human Fc를 fusion한 마우스 EphA10)만이 정제되었으며, 이의 항원으로서의 활성을 인간화 과정 전의 EphA10 특이적 마우스 항체 (m#2)를 이용한 ELISA 분석으로 확인하였다 (도 23). Since human EphA10 and mouse EphA10 have 95% similarity, if an antibody binds to mouse EphA10, it can be used as a good indicator for selecting a mouse model before in vivo experiments with EphA10-targeting antibodies. Therefore, an animal cell expression vector capable of expressing the extracellular domain (ECD) of mouse EphA10 together with various fusion proteins was prepared, transfected into Expi293F cells, and cultured for 7 days. Then, centrifugation was performed and the supernatant was collected. After adding 1/25 volume of 25× PBS, the supernatant was filtered using a 0.2 μm bottle top filter (Merck Millipore), and 1 ml of GSH resin, Protein A resin, or Ni-NTA resin was added to the filtered supernatant, and the mixture was stirred at 4 °C for 16 hours. After that, the mixture was passed through a column to recover each resin. After washing with 5 ml of 1× PBS, the solution was eluted with 50 mM Tris-HCl (pH 8.0), 200 mM glycine (pH 2.7), or 250 mM imidazole containing 4 ml of 10 mM reduced glutathione, respectively. After buffer replacement with 1× PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of the four purified antigen proteins were analyzed through SDS-PAGE. As a result of analyzing the size and purity of the four antigen proteins, only mEphA10-Fc (mouse EphA10 fused to human Fc) was purified among the four antigen proteins, and its activity as an antigen was confirmed by ELISA analysis using an EphA10-specific mouse antibody (m#2) before the humanization process (Fig. 23).

19-2. EphA10에 대한 결합력이 향상된 인간화 항체의 마우스 EphA10에 대한 결합력 분석19-2. Analysis of binding affinity of humanized antibodies with enhanced binding affinity to EphA10 to mouse EphA10

선발된 EphA10 특이적 인간화 항체 2종 (A.7 및 2-W)이 상기 실시예 19-1에서 발현 및 정제한 마우스 EphA10에도 결합하는지 확인하기 위해 ELISA를 진행하였다. 구체적으로, mEphA10-Fc 4μg/ml를 0.05 M Na2CO3 (pH 9.6) 용액으로 희석하여 50 μl씩 Flat Bottom Polystyrene High Bind 96 웰 마이크로플레이트 (costar)에 4℃에서 16시간 동안 고정화한 후, 100 μl의 4% 스킴밀크 (GenomicBase)가 포함된 PBS로 상온에서 2 시간 동안 블로킹하였다. 180 μl의 PBST로 4 회씩 세척한 뒤, 1% 스킴밀크에 연속 희석된 EPHA10 결합 항체들 (A.7 및 2-W)을 각각 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 세척 후 goat 항-human kappa chain HRP conjugate (Millipore) 50 μl씩을 첨가하여 상온에서 1 시간 동안 인큐베이션하고, 세척하였다. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific)을 50 μl씩 첨가하여 발색한 뒤 2 M의 H2SO4를 50 μl씩 넣어 반응을 종료시킨 후 Epoch Microplate Spectrophotometer (BioTek)를 이용하여 분석하였다. 그 결과, A7 항체 (A.7) 및 2W 항체 (2-W) 모두 마우스 EphA10에도 결합하는 것을 확인하였다 (도 24). To confirm whether the two selected EphA10-specific humanized antibodies (A.7 and 2-W) also bind to the mouse EphA10 expressed and purified in Example 19-1, ELISA was performed. Specifically, 4 μg/ml of mEphA10-Fc was diluted with 0.05 M Na 2 CO 3 (pH 9.6) solution, 50 μl each was immobilized on a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4°C for 16 hours, and then blocked with 100 μl of PBS containing 4% skim milk (GenomicBase) at room temperature for 2 hours. After washing four times with 180 μl of PBST, 50 μl of EPHA10-binding antibodies (A.7 and 2-W) serially diluted in 1% skim milk were dispensed into each well and reacted for 1 hour at room temperature. After washing, 50 μl of goat anti-human kappa chain HRP conjugate (Millipore) was added, incubated for 1 hour at room temperature, and washed. 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and 50 μl of 2 M H 2 SO 4 was added to terminate the reaction. The mixture was then analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, it was confirmed that both A7 antibody (A.7) and 2W antibody (2-W) bound to mouse EphA10 (Fig. 24).

Claims (38)

EphA10(Eph receptor A10)에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.An antibody specific for EphA10 (Eph receptor A10) or a fragment thereof having immunological activity. 제 1항에 있어서, 서열번호 1 내지 3의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRH(Complementarity determining regions Heavy chain)1, 서열번호 4 내지 6의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRH2, 및 서열번호 7 또는 8의 아미노산 서열을 포함하는 CDRH3를 포함하는 VH 도메인을 포함하는, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.An antibody specific for EphA10 or a fragment thereof having immunological activity, comprising a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 3, CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 4 to 6, and CDRH3 comprising an amino acid sequence of SEQ ID NO: 7 or 8. 제 1항에 있어서, 서열번호 9 내지 14의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRL1, 서열번호 15 또는 16의 아미노산 서열을 포함하는 CDRL2, 및 서열번호 17 내지 19의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 CDRL3를 포함하는 VL 도메인을 포함하는, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.An antibody specific for EphA10 or a fragment thereof having immunological activity, comprising a VL domain comprising CDRL1 selected from the group consisting of amino acid sequences of SEQ ID NOs: 9 to 14, CDRL2 comprising the amino acid sequence of SEQ ID NOs: 15 or 16, and CDRL3 selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19. 제 1항에 있어서, 서열번호 20 내지 25의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 VH 도메인을 포함하는, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.An antibody specific for EphA10 or a fragment thereof having immunological activity, comprising a VH domain selected from the group consisting of amino acid sequences of SEQ ID NOs: 20 to 25, in claim 1. 제 1항에 있어서, 서열번호 26 내지 45의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 VL 도메인을 포함하는, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.An antibody specific for EphA10 or a fragment thereof having immunological activity, comprising a VL domain selected from the group consisting of amino acid sequences of SEQ ID NOs: 26 to 45, in claim 1. 제 1항에 있어서, 면역학적 활성을 가진 단편은 Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv(single chain fragment variable), Fv, 단일쇄 항체, Fv 이량체, 상보성 결정 영역 단편, 인간화 항체, 키메라 항체 및 디아바디(diabody)로 이루어진 군으로부터 선택되는 어느 하나인, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.In claim 1, the fragment having immunological activity is any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody, an antibody specific for EphA10 or a fragment thereof having immunological activity. 제 1항에 있어서, 인간화 항체인, EphA10에 특이적인 항체 또는 이의 면역학적 활성을 가진 단편.In claim 1, an antibody specific for EphA10, which is a humanized antibody, or a fragment thereof having immunological activity. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 및 약물을 포함하는, 항체-약물 접합체(Antibody-Drug Conjugate, ADC).An antibody-drug conjugate (ADC) comprising an antibody of claim 1 or a fragment thereof having immunological activity, and a drug. 제 8항에 있어서, 약물은 면역원성 세포사멸 유도제, 전세포사멸성(pro-apoptotic) 펩타이드, 이크로튜불린(microtubulin) 구조 형성 억제제, 유사분열(meiosis) 억제제, 토포아이소머라아제(topoisomerase) 억제제, DNA 인터컬레이터(DNA intercalators), 독소(toxin), 방사선 핵종 또는 항암제인, 항체-약물 접합체.An antibody-drug conjugate in claim 8, wherein the drug is an immunogenic apoptosis inducer, a pro-apoptotic peptide, a microtubulin structure formation inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, a DNA intercalator, a toxin, a radionuclide or an anticancer agent. 제 9항에 있어서, 약물은 SN-38(7-에틸-10-히드록시-캠토테신, 7-Ethyl-10-hydroxy-camptothecin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin), 발루비신(valrubicin), 파클리탁셀(paclitaxel), 도세탁셀(docetaxel), 메클로에타민(mechloethamine), 클로람부실(chlorambucil), 페닐알라닌(phenylalanine), 무스타드(mustard), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 카르무스틴(carmustine: BCNU), 로무스틴(lomustine: CCNU), 스트렙토조토신(streptozotocin), 부설판(busulfan), 티오테파(thiotepa), 시스플라틴(cisplatin), 카보플라틴(carboplatin), 닥티노마이신(dactinomycin: actinomycin D), 플리카마이신(plicamycin), 마이토마이신 C(mitomycin C), 빈크리스틴(vincristine), 빈블라스틴(vinblastine), 테니포사이드(teniposide), 토포테칸(topotecan), 이리도테칸(iridotecan), 우라무스틴(uramustine), 멜파란(melphalan), 벤다무스틴(bendamustine), 다카바진(dacarbazine), 테모졸로마이드(temozolomide), 알트레타민(altretamine), 듀오카르마이신(duocarmycin), 네다플라틴(nedaplatin), 옥사리플라틴(oxaliplatin), 사트라플라틴(satraplatin), 트리플라틴 테트라나이트레이트(triplatin tetranitrate), 5-플루오로우라실(5-fluorouracil), 6-머캅토퓨린(6-mercaptopurine), 카페시타빈(capecitabine), 클라드리빈(cladribine), 클로파라빈(clofarabine), 시스타르빈(cystarbine), 플록스유리딘(floxuridine), 플루다라빈(fludarabine), 겜시타빈(gemcitabine), 하이드록시우레아(hydroxyurea), 메토트렉세이트(methotrexate), 페메트렉세드(pemetrexed), 펜토스타틴(pentostatin), 티오구아닌(thioguanine), 에토포사이드(etoposide), 미토산트론(mitoxantrone), 이자베필론(izabepilone), 빈데신(vindesine), 비노렐빈(vinorelbine), 에스트라머스틴(estramustine), 메이탄신(maytansine), DM1(mertansine, 메르탄신), DM4, 돌라스타틴(dolastatin), 아우리스타틴 E(auristatin E), 아우리스타틴 F(auristatin F), 모노메틸 아우리스타틴 E(monomethyl auristatin E, MMAE), 모노메틸 아우리스타틴 F(monomethyl auristatin F) 및 이들의 유도체로 이루어진 군으로부터 선택되는, 항체-약물 접합체.In claim 9, the drug is SN-38 (7-Ethyl-10-hydroxy-camptothecin), daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, valrubicin, paclitaxel, docetaxel, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), Streptozotocin, busulfan, thiotepa, cisplatin, carboplatin, dactinomycin (actinomycin D), plicamycin, mitomycin C, vincristine, vinblastine, teniposide, topotecan, iridotecan, uramustine, melphalan, bendamustine, dacarbazine, temozolomide, altretamine, duocarmycin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, etoposide, mitoxantrone, izabepilone, vindesine, vinorelbine, estramustine, An antibody-drug conjugate selected from the group consisting of maytansine, DM1 (mertansine), DM4, dolastatin, auristatin E, auristatin F, monomethyl auristatin E (MMAE), monomethyl auristatin F, and derivatives thereof. 제 8항에 있어서, ADC 링커를 추가로 포함하는, 항체-약물 접합체.An antibody-drug conjugate further comprising an ADC linker in claim 8. 제 11항에 있어서, ADC 링커는 6-말레이미도카프로일(MC), 말레이미도프로파노일(MP), 발린-시트룰린(val-cit), 알라닌-페닐알라닌(ala-phe), p-아미노벤질옥시카르보닐(PAB), N-숙신이미딜 4-(2-피리딜티오) 펜타노에이트(SPP), N-숙신이미딜 4-(N-말레이미도메틸) 시클로헥산-1 카르복실레이트(SMCC), 발린-시트룰린-p-아미노벤질옥시카르보닐(val-cit-PAB) 또는 N-숙신이미딜 (4-요오도-아세틸) 아미노벤조에이트(SIAB)인, 항체-약물 접합체.An antibody-drug conjugate in claim 11, wherein the ADC linker is 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), valine-citrulline-p-aminobenzyloxycarbonyl (val-cit-PAB), or N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB). 제 11항에 있어서, ADC 링커를 통해 항체 또는 이의 면역학적 활성을 가진 단편이 약물과 복합체를 형성하는, 항체-약물 접합체.An antibody-drug conjugate in claim 11, wherein an antibody or a fragment thereof having immunological activity forms a complex with a drug through an ADC linker. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 및 EphA10 이외의 표적 항원에 결합하는 부분을 포함하는 이중특이적 또는 다중특이적 항체. A bispecific or multispecific antibody comprising the antibody of claim 1 or a fragment having immunological activity thereof, and a portion that binds to a target antigen other than EphA10. 제 14항에 있어서, 표적 항원에 결합하는 부분이 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는, 이중특이적 또는 다중특이적 항체.A bispecific or multispecific antibody in claim 14, wherein the portion binding to the target antigen comprises an antibody or a fragment having immunological activity thereof. 제 14항에 있어서, 표적 항원은 17-1A 항원, GD3 갱글리오시드 R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 코어 단백질, 이상 글리코실화 MUC1, ED-B 도메인을 함유하는 피브로넥틴 이형, HER2/neu, 암종배아성 항원(CEA), 가스트린-방출 펩티드(GRP) 수용체 항원, 뮤신(mucine) 항원, 표피 성장 인자 수용체(EGF-R), HER3, HER4, MAGE 항원, SART 항원, MUC1 항원, c-erb-2 항원, TAG 72, 탄산 무수화효소 IX(carbonic anhydrase IX), 알파-태아단백질(alpha-fetoprotein), A3, A33 항체에 특이적인 항원, Ba 733, BrE3-항원, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, 결장-특이적 항원-p(CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, 폴산염 수용체(folate receptor), HLA-DR, 인간 융모성 성선자극호르몬(HCG) 및 그 소단위체들, 저산소증 유도 인자(HIF-I), Ia, IL-2, IL-6, IL-8, 인슐린 성장 인자-1(IGF-1), KC4-항원, KS-1-항원, KSl-4, Le-Y, 대식세포 억제 인자(MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM-4 항체에 특이적인 항원, 태반 성장 인자, p53, 전립선 산성 포스파타제(prostatic acid phosphatase), PSA, RS5, SlOO, TAC, 테나신(tenascin), TRAIL 수용체들, Tn 항원, 톰슨-프리덴라이히(Thomson-Friedenreich) 항원들, 종양 괴사 항원들, VEGF, ED-B 피브로넥틴(fibronectin), 혈관형성 표지(angiogenesis marker), 종양유전자 표지(oncogene marker) 또는 종양유전자 생성물로 이루어지는 군으로부터 선택되는 어느 하나 이상인, 이중특이적 또는 다중특이적 항체.In claim 14, the target antigen is an antigen specific for 17-1A antigen, GD3 ganglioside R24, EGFRvⅢ, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrantly glycosylated MUC1, fibronectin isoform containing ED-B domain, HER2/neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucin antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, A33 antibody, Ba 733, BrE3-antigen, CA125, CD1, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KSl-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, A bispecific or multispecific antibody, wherein the antibody is at least one selected from the group consisting of antigens specific for NCA66, NCA95, NCA90, PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase, PSA, RS5, SlOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, angiogenesis marker, oncogene marker or oncogene product. 제 14항에 있어서, 표적 항원은 세포 표면 항원 또는 자가항원인, 이중특이적 또는 다중특이적 항체.In claim 14, a bispecific or multispecific antibody, wherein the target antigen is a cell surface antigen or an autoantigen. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편 또는 제 14항의 이중특이적 또는 다중특이적 항체를 코딩하는 단리된 핵산 분자.An isolated nucleic acid molecule encoding an antibody of claim 1 or a fragment thereof having immunological activity or a bispecific or multispecific antibody of claim 14. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 또는 제 14항의 이중특이적 또는 다중특이적 항체를 유효성분으로 포함하는 자가면역 질환, 퇴행성신경 질환(neurodegerative disease), 알츠하이머병, 대사 질환(metabolic disease), 심혈관 질환(cardiovascular disease), 죽상동맥경화증(atherosclerosis), 기관 이식 거부반응, 진균, 바이러스, 박테리아 또는 기생충에 의하여 유발되는 질환 또는 증상으로 이루어진 군으로부터 선택되는 질병 또는 질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of a disease or condition selected from the group consisting of autoimmune diseases, neurodegenerative diseases, Alzheimer's disease, metabolic diseases, cardiovascular diseases, atherosclerosis, organ transplant rejection, diseases or symptoms caused by fungi, viruses, bacteria or parasites, comprising the antibody of claim 1 or a fragment having immunological activity thereof, the antibody-drug conjugate of claim 8, or the bispecific or multispecific antibody of claim 14 as an active ingredient. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편을 항원 결합 도메인으로 포함하는 키메라 항원 수용체(Chimeric antigen receptor: CAR).A chimeric antigen receptor (CAR) comprising an antibody of claim 1 or a fragment thereof having immunological activity as an antigen-binding domain. 제 20항의 키메라 항원 수용체를 암호화하는 유전자를 포함하는 재조합 벡터.A recombinant vector comprising a gene encoding a chimeric antigen receptor of claim 20. 제 21항의 재조합 벡터로 형질전환된 키메라 항원 수용체 발현 세포.A chimeric antigen receptor expressing cell transformed with the recombinant vector of claim 21. 제 22항에 있어서, 키메라 항원 수용체 발현 세포는 키메라 항원 수용체 발현 T(CAR-T) 세포 또는 자연살해(CAR-NK) 세포인, 키메라 항원 수용체 발현 세포.In claim 22, the chimeric antigen receptor expressing cell is a chimeric antigen receptor expressing T (CAR-T) cell or a natural killer (CAR-NK) cell. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편을 포함하는 T 세포 관여자(T-cell engager).A T-cell engager comprising an antibody of claim 1 or a fragment thereof having immunological activity. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 20항의 키메라 항원 수용체, 제 22항의 키메라 항원 수용체 발현 세포 또는 제 24항의 T 세포 관여자를 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an antibody of claim 1 or a fragment thereof having immunological activity, an antibody-drug conjugate of claim 8, a bispecific or multispecific antibody of claim 14, a chimeric antigen receptor of claim 20, a chimeric antigen receptor expressing cell of claim 22, or a T cell engager of claim 24. 제 25항에 있어서, 암은 뇌종양, 흑색종, 골수종, 비소세포성폐암, 구강암, 간암, 위암, 결장암, 유방암, 삼중음성유방암(Triple Negative Breast Cancer, TNBC), 폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 나팔관암종, 항문부근암, 자궁내막암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나인, 암의 예방 또는 치료용 약학적 조성물.In claim 25, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer (TNBC), lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, A pharmaceutical composition for the prevention or treatment of cancer, wherein the composition is any one selected from the group consisting of brainstem glioma and pituitary adenoma. 제 25항에 있어서, 암은 면역관문 억제제에 대해 불응성 암인, 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, wherein the cancer is refractory to an immune checkpoint inhibitor, in claim 25. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 20항의 키메라 항원 수용체, 제 22항의 키메라 항원 수용체 발현 세포, 또는 제 24항의 T 세포 관여자를 유효성분으로 포함하는 항암 보조제.An anticancer adjuvant comprising, as an active ingredient, the antibody of claim 1 or a fragment thereof having immunological activity, the antibody-drug conjugate of claim 8, the bispecific or multispecific antibody of claim 14, the chimeric antigen receptor of claim 20, the chimeric antigen receptor expressing cell of claim 22, or the T cell engager of claim 24. 제 28항에 있어서, 면역항암제와 동시에, 별도로, 또는 순차적으로 병용 투여되는 항암 보조제.In claim 28, an anticancer adjuvant agent administered concurrently, separately, or sequentially with an immunotherapy agent. 제 28항에 있어서, 면역항암제에 대한 불응성을 개선시키는 항암 보조제.An anticancer adjuvant that improves refractoriness to immunotherapy in claim 28. 제 28항에 있어서, 면역항암제는 면역관문 억제제(Immune checkpoint inhibitors), 면역억제인자 제어 약물, 암 백신, 면역아주번트(immunoadjuvant), 암 치료용 면역세포, 면역세포 활성 보조인자, 암 치료용 항체 또는 암 치료용 면역세포의 활성 유지에 필요한 사이토카인인, 항암 보조제.In claim 28, the immunotherapy agent is an anticancer adjuvant that is an immune checkpoint inhibitor, an immunosuppressant controlling drug, a cancer vaccine, an immunoadjuvant, an immune cell for cancer treatment, an immune cell activation cofactor, an antibody for cancer treatment, or a cytokine necessary for maintaining the activity of an immune cell for cancer treatment. 제 31항에 있어서, 면역관문 억제제는 CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA 또는 A2aR의 억제제인, 항암 보조제.In claim 31, the immune checkpoint inhibitor is an anticancer adjuvant agent which is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, TIGIT, CD47, VISTA or A2aR. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 20항의 키메라 항원 수용체, 제 22항의 키메라 항원 수용체 발현 세포, 또는 제 24항의 T 세포 관여자를 유효성분으로 포함하는 암 진단용 조성물.A composition for diagnosing cancer, comprising as an active ingredient an antibody of claim 1 or a fragment thereof having immunological activity, an antibody-drug conjugate of claim 8, a bispecific or multispecific antibody of claim 14, a chimeric antigen receptor of claim 20, a chimeric antigen receptor expressing cell of claim 22, or a T cell engager of claim 24. 제 33항에 있어서, 표지를 추가로 포함하는, 암 진단용 조성물.A composition for diagnosing cancer, further comprising a label in claim 33. 제 34항에 있어서, 표지는 발색효소, 방사성 동위원소, 크로모포어(chromopore), 발광물질, 형광물질, 프로브 또는 택(tag)인, 암 진단용 조성물.A composition for diagnosing cancer, wherein the label is a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, a fluorescent substance, a probe or a tag in claim 34. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 22항의 키메라 항원 수용체 발현 세포, 또는 제 24항의 T 세포 관여자의 암의 예방 또는 치료 용도.Use of the antibody of claim 1 or a fragment thereof having immunological activity, the antibody-drug conjugate of claim 8, the bispecific or multispecific antibody of claim 14, the chimeric antigen receptor expressing cell of claim 22, or the T cell engager of claim 24 for the prevention or treatment of cancer. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 22항의 키메라 항원 수용체 발현 세포, 또는 제 24항의 T 세포 관여자를 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암의 치료 방법.A method for treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the antibody of claim 1 or a fragment thereof having immunological activity, the antibody-drug conjugate of claim 8, the bispecific or multispecific antibody of claim 14, the chimeric antigen receptor expressing cell of claim 22, or the T cell engager of claim 24. 제 1항의 항체 또는 이의 면역학적 활성을 가진 단편, 제 8항의 항체-약물 접합체, 제 14항의 이중특이적 또는 다중특이적 항체, 제 20항의 키메라 항원 수용체, 제 22항의 키메라 항원 수용체 발현 세포, 또는 제 24항의 T 세포 관여자의 암 진단 용도.Use of the antibody of claim 1 or a fragment thereof having immunological activity, the antibody-drug conjugate of claim 8, the bispecific or multispecific antibody of claim 14, the chimeric antigen receptor of claim 20, the chimeric antigen receptor expressing cell of claim 22, or the T cell engager of claim 24 for the diagnosis of cancer.
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Title
CHA JONG-HO, CHAN LI-CHUAN, WANG YING-NAI, CHU YU-YI, WANG CHIE-HONG, LEE HENG-HUAN, XIA WEIYA, SHYU WOEI-CHERNG, LIU SHIH-PING, Y: "Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 298, no. 4, 1 April 2022 (2022-04-01), US , pages 1 - 16, XP093216512, ISSN: 0021-9258, DOI: 10.1016/j.jbc.2022.101817 *
NAGANO KAZUYA; MAEDA YUKA; KANASAKI SO-ICHIRO; WATANABE TAKANOBU; YAMASHITA TAKUYA; INOUE MASAKI; HIGASHISAKA KAZUMA; YOSHIOKA YAS: "Ephrin receptor A10 is a promising drug target potentially useful for breast cancers including triple negative breast cancers", JOURNAL OF CONTROLLED RELEASE, vol. 189, 16 June 2014 (2014-06-16), AMSTERDAM, NL , pages 72 - 79, XP029015087, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2014.06.010 *

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